TARGETED TGFß INHIBITION

ABSTRACT

This invention relates generally to bifunctional molecules including (a) a TGFβRII or fragment thereof capable of binding TGFβ and (b) an antibody, or antigen binding fragment thereof, that binds to an immune checkpoint protein, such as Programmed Death Ligand 1 (PD-L1), uses of such molecules (e.g., for treating cancer), and methods of making such molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to pending U.S. Provisional PatentApplication No. 61/938,048, filed on Feb. 10, 2014, entitled “TargetedTGFβ Inhibition,” the disclosure of which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to bifunctional molecules including (a)a TGFβRII or fragment thereof capable of binding TGFβ and (b) anantibody, or antigen binding fragment thereof, that binds to an immunecheckpoint protein, such as Programmed Death Ligand 1 (PD-L1), uses ofsuch molecules (e.g., for treating cancer), and methods of making suchmolecules.

BACKGROUND

In cancer treatment, it has long been recognized that chemotherapy isassociated with high toxicity and can lead to emergence of resistantcancer cell variants. Even with targeted therapy against overexpressedor activated oncoproteins important for tumor survival and growth,cancer cells invariably mutate and adapt to reduce dependency on thetargeted pathway, such as by utilizing a redundant pathway. Cancerimmunotherapy is a new paradigm in cancer treatment that instead oftargeting cancer cells, focuses on the activation of the immune system.Its principle is to rearm the host's immune response, especially theadaptive T cell response, to provide immune surveillance to kill thecancer cells, in particular, the minimal residual disease that hasescaped other forms of treatment, hence achieving long-lastingprotective immunity.

FDA approval of the anti-CTLA-4 antibody ipilimumab for the treatment ofmelanoma in 2011 ushered in a new era of cancer immunotherapy. Thedemonstration that anti-PD-1 or anti-PD-L1 therapy induced durableresponses in melanoma, kidney, and lung cancer in clinical trialsfurther signify its coming of age (Pardoll, D. M., Nat Immunol. 2012;13:1129-32). However, ipilimumab therapy is limited by its toxicityprofile, presumably because anti-CTLA-4 treatment, by interfering withthe primary T cell inhibitory checkpoint, can lead to the generation ofnew autoreactive T cells. While inhibiting the PD-L1/PD-1 interactionresults in dis-inhibiting existing chronic immune responses in exhaustedT cells that are mostly antiviral or anticancer in nature (Wherry, E.J., Nat Immunol. 2011; 12:492-9), anti-PD-1 therapy can neverthelesssometimes result in potentially fatal lung-related autoimmune adverseevents. Despite the promising clinical activities of anti-PD1 andanti-PD-L1 so far, increasing the therapeutic index, either byincreasing therapeutic activity or decreasing toxicity, or both, remainsa central goal in the development of immunotherapeutics.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that a bifunctionalprotein containing at least portion of TGFβ Receptor II (TGFβRII) thatis capable of binding TGFβ and antibody or antigen-binding fragment thatbinds to an immune checkpoint protein such as human protein ProgrammedDeath Ligand 1 (PD-L1) can be an effective anti-tumor and anti-cancertherapeutic. The protein can exhibit a synergistic effect in cancertreatment, as compared to the effect of administering the two agentsseparately.

Accordingly, in a first aspect, the present invention features a proteinincluding (a) human TGFβRII, or a fragment thereof capable of bindingTGFβ (e.g., a soluble fragment); and (b) an antibody, or anantigen-binding fragment thereof, that binds PD-L1 (e.g., any of theantibodies or antibody fragments described herein).

In a related aspect, the invention features a polypeptide including (a)at least a variable domain of a heavy chain of an antibody that bindsPD-L1 (e.g., amino acids 1-120 of SEQ ID NO: 2); and (b) human TGFβRII,or a soluble fragment thereof capable of binding TGFβ (e.g., a humanTGFβRII extra-cellular domain (ECD), amino acids 24-159 of SEQ ID NO: 9,or any of those described herein). The polypeptide may further includean amino acid linker connecting the C-terminus of the variable domain tothe N-terminus of the human TGFβRII or soluble fragment thereof capableof binding TGFβ. The polypeptide may include the amino acid sequence ofSEQ ID NO: 3 or an amino acid sequence substantially identical to SEQ IDNO: 3. The antibody fragment may be an scFv, Fab, F(ab′)₂, or Fvfragment.

In certain embodiments, the protein or polypeptide includes an antibodyor antigen-binding fragment thereof that includes SEQ ID NO: 2 and humanTGFβRII. The antibody may optionally include a modified constant region(e.g., any described herein, including a C-terminal Lys→Alasubstitution, a mutation of the Leu-Ser-Leu-Ser (SEQ ID NO: 19) sequenceto Ala-Thr-Ala-Thr (SEQ ID NO: 20), or a hybrid constant regionincluding an IgG1 hinge region and an IgG2 CH2 domain).

In certain embodiments, the protein or polypeptide includes an antibodyor antigen-binding fragment thereof that includes SEQ ID NO: 2 and afragment of human TGFβRII capable of binding TGFβ (e.g., a solublefragment). The antibody may optionally include a modified constantregion (e.g., any described herein, including a C-terminal Lys→Alasubstitution, a mutation of the Leu-Ser-Leu-Ser (SEQ ID NO: 19) sequenceto Ala-Thr-Ala-Thr (SEQ ID NO: 20), or a hybrid constant regionincluding an IgG1 hinge region and an IgG2 CH2 domain).

In certain embodiments, the protein or polypeptide includes an antibodyor antigen-binding fragment thereof that includes SEQ ID NO: 2 and ahuman TGFβRII ECD. The antibody may include a modified constant region(e.g., any described herein, including a C-terminal Lys→Alasubstitution, a mutation of the Leu-Ser-Leu-Ser (SEQ ID NO: 19) sequenceto Ala-Thr-Ala-Thr (SEQ ID NO: 20), or a hybrid constant regionincluding an IgG1 hinge region and an IgG2 CH2 domain).

In certain embodiments, the protein or polypeptide includes an antibodyor antigen-binding fragment thereof that includes amino acids 1-120 ofSEQ ID NO: 2 and human TGFβRII. The antibody may include a modifiedconstant region (e.g., any described herein, including a C-terminalLys→Ala substitution, a mutation of the Leu-Ser-Leu-Ser (SEQ ID NO: 19)sequence to Ala-Thr-Ala-Thr (SEQ ID NO: 20), or a hybrid constant regionincluding an IgG1 hinge region and an IgG2 CH2 domain).

In certain embodiments, the protein or polypeptide includes an antibodyor antigen-binding fragment thereof that includes amino acids 1-120 ofSEQ ID NO: 2 and a fragment of human TGFβRII capable of binding TGFβ(e.g., a soluble fragment). The antibody may include a modified constantregion (e.g., any described herein, including a C-terminal Lys→Alasubstitution, a mutation of the Leu-Ser-Leu-Ser (SEQ ID NO: 19) sequenceto Ala-Thr-Ala-Thr (SEQ ID NO: 20), or a hybrid constant regionincluding an IgG1 hinge region and an IgG2 CH2 domain).

In certain embodiments, the protein or polypeptide includes an antibodyor antigen-binding fragment thereof that includes amino acids 1-120 ofSEQ ID NO: 2 and a human TGFβRII ECD. The antibody may include amodified constant region (e.g., any described herein, including aC-terminal Lys→Ala substitution, a mutation of the Leu-Ser-Leu-Ser (SEQID NO: 19) sequence to Ala-Thr-Ala-Thr (SEQ ID NO: 20), or a hybridconstant region including an IgG1 hinge region and an IgG2 CH2 domain).

In certain embodiments, the protein or polypeptide includes an antibodyor antigen-binding fragment thereof that includes the hypervariableregions present in SEQ ID NO: 2 and human TGFβRII. The antibody mayinclude a modified constant region (e.g., any described herein,including a C-terminal Lys→Ala substitution, a mutation of theLeu-Ser-Leu-Ser (SEQ ID NO: 19) sequence to Ala-Thr-Ala-Thr (SEQ ID NO:20), or a hybrid constant region including an IgG1 hinge region and anIgG2 CH2 domain).

In certain embodiments, the protein or polypeptide includes an antibodyor antigen-binding fragment thereof that includes the hypervariableregions present in SEQ ID NO: 2 and a fragment of human TGFβRII capableof binding TGFβ (e.g., a soluble fragment). The antibody may include amodified constant region (e.g., any described herein, including aC-terminal Lys→Ala substitution, a mutation of the Leu-Ser-Leu-Ser (SEQID NO: 19) sequence to Ala-Thr-Ala-Thr (SEQ ID NO: 20), or a hybridconstant region including an IgG1 hinge region and an IgG2 CH2 domain).

In certain embodiments, the protein or polypeptide includes an antibodyor antigen-binding fragment thereof that includes the hypervariableregions present in SEQ ID NO: 2 and a human TGFβRII ECD. The antibodymay include a modified constant region (e.g., any described herein,including a C-terminal Lys→Ala substitution, a mutation of theLeu-Ser-Leu-Ser (SEQ ID NO: 19) sequence to Ala-Thr-Ala-Thr (SEQ ID NO:20), or a hybrid constant region including an IgG1 hinge region and anIgG2 CH2 domain).

In certain embodiments, the protein or polypeptide includes an antibodyor antigen-binding fragment thereof that includes SEQ ID NO: 12 andhuman TGFβRII. The antibody may include a modified constant region(e.g., any described herein, including a C-terminal Lys→Alasubstitution, a mutation of the Leu-Ser-Leu-Ser (SEQ ID NO: 19) sequenceto Ala-Thr-Ala-Thr (SEQ ID NO: 20), or a hybrid constant regionincluding an IgG1 hinge region and an IgG2 CH2 domain).

In certain embodiments, the protein or polypeptide includes an antibodyor antigen-binding fragment thereof that includes SEQ ID NO: 12 and afragment of human TGFβRII capable of binding TGFβ (e.g., a solublefragment). The antibody may include a modified constant region (e.g.,any described herein, including a C-terminal Lys→Ala substitution, amutation of the Leu-Ser-Leu-Ser (SEQ ID NO: 19) sequence toAla-Thr-Ala-Thr (SEQ ID NO: 20), or a hybrid constant region includingan IgG1 hinge region and an IgG2 CH2 domain).

In certain embodiments, the protein or polypeptide includes an antibodyor antigen-binding fragment thereof that includes SEQ ID NO: 12 and ahuman TGFβRII ECD. The antibody may include a modified constant region(e.g., any described herein, including a C-terminal Lys→Alasubstitution, a mutation of the Leu-Ser-Leu-Ser (SEQ ID NO: 19) sequenceto Ala-Thr-Ala-Thr (SEQ ID NO: 20), or a hybrid constant regionincluding an IgG1 hinge region and an IgG2 CH2 domain).

In certain embodiments, the protein or polypeptide includes an antibodyor antigen-binding fragment thereof that includes the hypervariableregions present in SEQ ID NO: 12 and human TGFβRII. The antibody mayinclude a modified constant region (e.g., any described herein,including a C-terminal Lys→Ala substitution, a mutation of theLeu-Ser-Leu-Ser (SEQ ID NO: 19) sequence to Ala-Thr-Ala-Thr (SEQ ID NO:20), or a hybrid constant region including an IgG1 hinge region and anIgG2 CH2 domain).

In certain embodiments, the protein or polypeptide includes an antibodyor antigen-binding fragment thereof that includes the hypervariableregions present in SEQ ID NO: 12 and a fragment of human TGFβRII capableof binding TGFβ (e.g., a soluble fragment). The antibody may include amodified constant region (e.g., any described herein, including aC-terminal Lys→Ala substitution, a mutation of the Leu-Ser-Leu-Ser (SEQID NO: 19) sequence to Ala-Thr-Ala-Thr (SEQ ID NO: 20), or a hybridconstant region including an IgG1 hinge region and an IgG2 CH2 domain).

In certain embodiments, the protein or polypeptide includes an antibodyor antigen-binding fragment thereof that includes the hypervariableregions present in SEQ ID NO: 12 and a human TGFβRII ECD. The antibodymay include a modified constant region (e.g., any described herein,including a C-terminal Lys→Ala substitution, a mutation of theLeu-Ser-Leu-Ser (SEQ ID NO: 19) sequence to Ala-Thr-Ala-Thr (SEQ ID NO:20), or a hybrid constant region including an IgG1 hinge region and anIgG2 CH2 domain).

In certain embodiments, the protein or polypeptide includes an antibodyor antigen-binding fragment thereof that includes SEQ ID NO: 14 andhuman TGFβRII. The antibody may include a modified constant region(e.g., any described herein, including a C-terminal Lys→Alasubstitution, a mutation of the Leu-Ser-Leu-Ser (SEQ ID NO: 19) sequenceto Ala-Thr-Ala-Thr (SEQ ID NO: 20), or a hybrid constant regionincluding an IgG1 hinge region and an IgG2 CH2 domain).

In certain embodiments, the protein or polypeptide includes an antibodyor antigen-binding fragment thereof that includes SEQ ID NO: 14 and afragment of human TGFβRII capable of binding TGFβ (e.g., a solublefragment). The antibody may include a modified constant region (e.g.,any described herein, including a C-terminal Lys→Ala substitution, amutation of the Leu-Ser-Leu-Ser (SEQ ID NO: 19) sequence toAla-Thr-Ala-Thr (SEQ ID NO: 20), or a hybrid constant region includingan IgG1 hinge region and an IgG2 CH2 domain).

In certain embodiments, the protein or polypeptide includes an antibodyor antigen-binding fragment thereof that includes SEQ ID NO: 14 and ahuman TGFβRII ECD. The antibody may include a modified constant region(e.g., any described herein, including a C-terminal Lys→Alasubstitution, a mutation of the Leu-Ser-Leu-Ser (SEQ ID NO: 19) sequenceto Ala-Thr-Ala-Thr (SEQ ID NO: 20), or a hybrid constant regionincluding an IgG1 hinge region and an IgG2 CH2 domain).

The invention also features a nucleic acid that includes a nucleotidesequence that encodes a polypeptide described above. In certainembodiments, the nucleic acid further includes a second nucleotidesequence encoding at least a variable domain of a light chain of anantibody which, when combined with the polypeptide, forms anantigen-binding site that binds PD-L1 (e.g., including amino acids 1-110of SEQ ID NO: 1). The second nucleotide sequence may encode the aminoacid sequence of SEQ ID NO: 1 (secreted anti-PD-L1 lambda light chain)or an amino acid sequence substantially identical to SEQ ID NO: 1. Theinvention also features a cell including any of the nucleic acidsdescribed above.

The invention also features a method of producing a protein including(a) the extracellular domain of the human TGFβRII, or a fragment thereofcapable of binding TGFβ (e.g., a soluble fragment), and (b) an antibody,or an antigen-binding fragment thereof, that binds human PD-L1. Themethod includes maintaining a cell described under conditions thatpermit expression of the protein. The method may further includeharvesting the protein.

The invention also features a protein including the polypeptidedescribed above and at least a variable domain of a light chain of anantibody which, when combined with the polypeptide, forms anantigen-binding site that binds PD-L1. The protein may include (a) twopolypeptides, each having an amino acid sequence consisting of the aminoacid sequence of SEQ ID NO: 3, and (b) two additional polypeptides eachhaving an amino acid sequence consisting of the amino acid sequence ofSEQ ID NO: 1.

The invention also features a protein described above for use intherapy. The therapy may include administration of radiation oradministration of a chemotherapeutic, a biologic, or a vaccine.

The invention also features a protein described above for use inpromoting local depletion of TGFβ at a tumor.

The invention also features a protein described above for use ininhibiting SMAD3 phosphorylation in a cell (e.g., a tumor cell or animmune cell).

The invention also features a protein described above for use intreating cancer or for use in inhibiting tumor growth. The cancer ortumor may be selected from the group consisting of colorectal, breast,ovarian, pancreatic, gastric, prostate, renal, cervical, myeloma,lymphoma, leukemia, thyroid, endometrial, uterine, bladder,neuroendocrine, head and neck, liver, nasopharyngeal, testicular, smallcell lung cancer, non-small cell lung cancer, melanoma, basal cell skincancer, squamous cell skin cancer, dermatofibrosarcoma protuberans,Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, andmyelodisplastic syndromes. The use may further include administration ofradiation or administration of a chemotherapeutic, a biologic, or avaccine.

The invention also features a method of promoting local depletion ofTGFβ. The method includes administering a protein described above, wherethe protein binds TGFβ in solution, binds PD-L1 on a cell surface, andcarries the bound TGFβ into the cell (e.g., a cancer cell).

The invention also features a method of inhibiting SMAD3 phosphorylationin a cell (e.g., a cancer cell or an immune cell), the method includingexposing the cell in the tumor microenvironment to a protein describedabove.

The invention also features a method of inhibiting tumor growth ortreating cancer. The method includes exposing the tumor to a proteindescribed above. The method may further include exposing the tumor toradiation or to a chemotherapeutic, a biologic, or a vaccine. In certainembodiments, the tumor or cancer is selected from the group consistingof colorectal, breast, ovarian, pancreatic, gastric, prostate, renal,cervical, myeloma, lymphoma, leukemia, thyroid, endometrial, uterine,bladder, neuroendocrine, head and neck, liver, nasopharyngeal,testicular, small cell lung cancer, non-small cell lung cancer,melanoma, basal cell skin cancer, squamous cell skin cancer,dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma,glioma, sarcoma, mesothelioma, and myelodisplastic syndromes.

By “TGFβRII” or “TGFβ Receptor II” is meant a polypeptide having thewild-type human TGFβ Receptor Type 2 Isoform A sequence (e.g., the aminoacid sequence of NCBI Reference Sequence (RefSeq) Accession No.NP_001020018 (SEQ ID NO: 8)), or a polypeptide having the wild-typehuman TGFβ Receptor Type 2 Isoform B sequence (e.g., the amino acidsequence of NCBI RefSeq Accession No. NP_003233 (SEQ ID NO: 9)) orhaving a sequence substantially identical the amino acid sequence of SEQID NO: 8 or of SEQ ID NO: 9. The TGFβRII may retain at least 0.1%, 0.5%,1%, 5%, 10%, 25%, 35%, 50%, 75%, 90%, 95%, or 99% of the TGFβ-bindingactivity of the wild-type sequence. The polypeptide of expressed TGFβRIIlacks the signal sequence.

By a “fragment of TGFβRII capable of binding TGFβ” is meant any portionof NCBI RefSeq Accession No. NP_001020018 (SEQ ID NO: 8) or of NCBIRefSeq Accession No. NP_003233 (SEQ ID NO: 9), or a sequencesubstantially identical to SEQ ID NO: 8 or SEQ ID NO: 9 that is at least20 (e.g., at least 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 175, or 200) amino acids in length that retains at least someof the TGFβ-binding activity (e.g., at least 0.1%, 0.5%, 1%, 5%, 10%,25%, 35%, 50%, 75%, 90%, 95%, or 99%) of the wild-type receptor or ofthe corresponding wild-type fragment. Typically such fragment is asoluble fragment. An exemplary such fragment is a TGFβRII extra-cellulardomain having the sequence of SEQ ID NO: 10.

By “substantially identical” is meant a polypeptide exhibiting at least50%, desirably 60%, 70%, 75%, or 80%, more desirably 85%, 90%, or 95%,and most desirably 99% amino acid sequence identity to a reference aminoacid sequence. The length of comparison sequences will generally be atleast 10 amino acids, desirably at least 15 contiguous amino acids, moredesirably at least 20, 25, 50, 75, 90, 100, 150, 200, 250, 300, or 350contiguous amino acids, and most desirably the full-length amino acidsequence.

By “patient” is meant either a human or non-human animal (e.g., amammal).

By “treating” a disease, disorder, or condition (e.g., a cancer) in apatient is meant reducing at least one symptom of the disease, disorder,or condition by administrating a therapeutic agent to the patient.

By “cancer” is meant a collection of cells multiplying in an abnormalmanner.

Other embodiments and details of the invention are presented hereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic drawing of an anti-PD-L1/TGFβ Trap moleculecomprising one anti-PD-L1 antibody fused to two extracellular domain(ECD) of TGFβ Receptor II via a (Gly₄Ser)₄Gly linker. FIG. 1B is aphotograph of a sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) analysis of anti-PD-L1/TGFβ Trap undernon-reducing and reducing conditions.

FIG. 2 is photograph of an SDS-PAGE gel showing analysis of extent ofclipping of anti-PD-L1/TGFβ Trap expressed by clone 02B15 at variouspopulation doubling levels. Anti-PD-L1/TGFβ Trap from clone 02B15 aftera single protein A chromatography step was analyzed by SDS-PAGE underreducing conditions. Lanes 1 and 10, See Blue Plus 2 MW Standard; lane2, purified anti-PD-L1/TGFβ Trap reference; lane 3, clone 02B15 at PDL0;lane 4, clone 02B15 at PDL30; lane 5, clone 02B15 at PDL60; and lane 6,clone 02B15 at PDL90. (PDL, population doubling level).

FIG. 3 is a graph showing FACS analysis of anti-PD-L1/TGFβ Trap bindingto HEK cells transfected to express human PD-L1.

FIG. 4 is a graph showing the ability of anti-PD-L1/TGFβ Trap to inhibitTGFβ-induced phosphorylation of SMAD3 using a pSMAD3-luciferase reportercell line (filled circle: anti-PD-L1; X: anti-PD-L1 (mut); filledsquare: anti-PD-L1/TGFβ Trap; filled triangle: anti-PD-L1(mut)/TGFβTrap; +: anti-TGFβ antibody 1D11; star: TGFβ RII-Fc).

FIGS. 5A and 5B are graphs showing pharmacokinetics of intravenouslyadministered anti-PD-L1/TGFβ Trap and related proteins in mice.

FIG. 6A is a graph showing PD-L1 target-mediated endocytosis ofanti-PD-L1/TGFβ Trap. FIG. 6B is a graph showing PD-L1 target-mediatedendocytosis of anti-PD-L1. FIG. 6C is a graph showing percentinternalization of anti-PD-L1/TGFβ Trap and anti-PD-L1 bound onHEK/PD-L1 cells.

FIGS. 7A-7C are graphs showing anti-tumor efficacy of anti-PD-L1/TGFβTrap and related proteins in the EMT-6 breast carcinoma subcutaneousmodel (Example 7). FIG. 7A shows tumor growth curves of average tumorvolumes of surviving mice in different treatment groups (star: Group 1:filled circle: Group 2; filled triangle: Group 3; filled square: Group4; open square: Group 5; filled square/dashed line: Group 6; filledsquare/stippled line: Group 7). FIG. 7B shows tumor growth curves ofindividual tumor volumes in different treatment groups. FIG. 7C is aKaplan-Meier plot of percent survival in different treatment groups(symbols as in 7A).

FIG. 8 is a graph showing anti-tumor efficacy of anti-PD-L1/TGFβ Trapand related proteins in the MC38 colorectal carcinoma subcutaneous tumormodel (Example 8; star: Group 1; filled circle: Group 2; filledcircle/dashed line: Group 3; filled triangle: Group 4; filledtriangle/dashed line: Group 5; filled square: Group 6; filledsquare/dashed line: Group 7).

FIG. 9 is a graph showing anti-tumor efficacy of anti-PDL1/TGFβ Trap andrelated proteins in an orthotopic EMT-6 breast cancer model (Example 9;star: Group 1; filled circle/dashed line: Group 2; filled triangle:Group 3; filled triangle/dashed line: Group 4; filled diamond: Group 5).

FIG. 10 is a graph showing anti-tumor efficacy of anti-PDL1/TGFβ Trapand related proteins in an intramuscular MC38 colorectal carcinoma model(Example 10; star: Group 1; filled circle: Group 2; filled circle/dashedline: Group 3: filled diamond/dashed line: Group 4; filled square: Group5; filled square/dashed line: Group 6; filled diamond: Group 7).

FIG. 11 is a graph showing anti-tumor efficacy of anti-PD-L1/TGF-β Trapand the combination of anti-PD-L1 and TGFβ Trap control administered togive equivalent in vivo exposure in an orthotopic EMT-6 breast tumormodel (Example 11; star: Group 1; filled square: Group 2; open square:Group 3; filled diamond: Group 4; open diamond: Group 5).

FIGS. 12A-12C are graphs showing anti-tumor efficacy of anti-PD-L1/TGF-βTrap and the combination of anti-PD-L1 and TGFβ Trap controladministered to give equivalent in vivo exposure in an intramuscularMC38 colorectal carcinoma model (Example 12). FIG. 12A shows tumorgrowth curves of mice treated with both intermediate and low doses ofthe proteins (star: Group 1; filled squares: Group 2; open squares:Group 3; filled diamonds: Group 4; open diamonds Group 5). FIG. 12B(star: Group 1; filled square: Group 2; filled diamond: Group 4; *:p<0.0001 compared to Group 1; **: p<0.0001 compared to Group 2) and 12C(star: Group 1; filled square: Group 3; filled diamond: Group 5; *:p<0.0001 compared to Group 1; **: p<0.0001 compared to Group 3) showstatistical analysis of tumor growth curves of mice treated withintermediate and low doses of the proteins, respectively

FIGS. 13A-13B are graphs showing anti-tumor efficacy ofanti-PD-L1(YW)/TGF-β Trap and related proteins in an orthotopic EMT-6breast tumor model (Example 13; star: Group 1; filled circle: Group 2;filled triangle: Group 3; filled square: Group 4; filled diamond: Group5). FIG. 13A shows tumor growth curves of mice in different treatmentgroups. FIG. 13B is a Kaplan-Meier plot of percent survival in differenttreatment groups.

FIGS. 14A-14B are graphs showing anti-tumor efficacy ofanti-PD-L1(YW)/TGF-β Trap and related proteins based on (A) tumorvolumes and (B) tumor weights, in an intramuscular MC38 colorectalcarcinoma model (Example 14; star: Group 1; filled circle: Group 2;filled triangle: Group 3; filled square: Group 4; filled diamond: Group5).

FIG. 15 is a graph comparing the anti-tumor efficacy of an anti-PD-1antibody treatment with and without TGFβ Trap control in an orthotopicEMT-6 breast tumor model (Example 15; star: Group 1; filled square:Group 2; filled inverted triangle: Group 3; open inverted triangle:Group 4).

FIG. 16 is a graph comparing the anti-tumor efficacy of an anti-PD-1antibody treatment with and without TGFβ Trap control in anintramuscular MC38 colorectal tumor model (Example 16; star: Group 1;filled square: Group 2; filled inverted triangle: Group 3; open invertedtriangle: Group 4).

FIG. 17 is a graph comparing the anti-tumor efficacy of an anti-LAG3 oranti-TIM3 antibody treatment with and without TGFβ Trap control in anorthotopic EMT-6 breast tumor model (Example 17; star: Group 1; filledsquare: Group 2; filled triangle: Group 3; filled inverted triangle:Group 4; open triangle: Group 5; open inverted triangle: Group 6).

FIG. 18 is a graph comparing the anti-tumor efficacy of an anti-LAG3 oranti-TIM3 antibody treatment with and without TGFβ Trap control in anintramuscular MC38 colorectal tumor model (Example 18; star: Group 1;filled square: Group 2; filled triangle: Group 3; filled invertedtriangle: Group 4; open triangle: Group 5; open inverted triangle: Group6).

DETAILED DESCRIPTION

The current invention permits localized reduction in TGFβ in a tumormicroenvironment by capturing the TGFβ using a soluble cytokine receptor(TGFβRII) tethered to an antibody moiety targeting a cellular immunecheckpoint receptor found on the exterior surface of certain tumor cellsor immune cells. An example of an antibody moiety of the invention to animmune checkpoint protein is anti-PD-L1. This bifunctional molecule,sometimes referred to in this document as an “antibody-cytokine trap,”is effective precisely because the anti-receptor antibody and cytokinetrap are physically linked. The resulting advantage (over, for example,administration of the antibody and the receptor as separate molecules)is partly because cytokines function predominantly in the localenvironment through autocrine and paracrine functions. The antibodymoiety directs the cytokine trap to the tumor microenvironment where itcan be most effective, by neutralizing the local immunosuppressiveautocrine or paracrine effects. Furthermore, in cases where the targetof the antibody is internalized upon antibody binding, an effectivemechanism for clearance of the cytokine/cytokine receptor complex isprovided. Antibody-mediated target internalization has been shown forPD-L1. This is a distinct advantage over using an anti-TGFβ antibodybecause first, an anti-TGFβ antibody might not be completelyneutralizing; and second, the antibody can act as a carrier extendingthe half-life of the cytokine, and antibody/cytokine complexes often actas a circulating sink that builds up and ultimately dissociates torelease the cytokine back in circulation (Montero-Julian et al., Blood.1995; 85:917-24). The use of a cytokine trap to neutralize the ligandcan also be a better strategy than blockading the receptor with anantibody, as in the case of CSF-1. Because CSF-1 is cleared from thecirculation by receptor-mediated endocytosis, an anti-CSF-1 receptorantibody blockade caused a significant increase in circulating CSF-1concentration (Hume et al., Blood. 2012; 119:1810-20)

Indeed, as described below, treatment with the anti-PD-L1/TGFβ Trapelicits a synergistic anti-tumor effect due to the simultaneous blockadeof the interaction between PD-L1 on tumor cells and PD-1 on immunecells, and the neutralization of TGFβ in the tumor microenvironment. Asdemonstrated in the following examples, anti-PDL1/TGFβ Trap has efficacysuperior to that of the single agent anti-PD-L1 or TGFβ Trap control.Without being bound by theory, this presumably is due to a synergisticeffect obtained from simultaneous blocking the two major immune escapemechanisms, and in addition, the targeted depletion of the TGFβ in thetumor microenvironment by a single molecular entity. This depletion isachieved by (1) anti-PD-L1 targeting of tumor cells; (2) binding of theTGFβ autocrine/paracrine in the tumor microenvironment by the TGFβ Trap;and (3) destruction of the bound TGFβ through the PD-L1receptor-mediated endocytosis. The aforementioned mechanisms of actioncannot be achieved by the combination therapy of the two single agentsanti-PD-L1 and TGFβ Trap. Furthermore, the TGFβRII fused to theC-terminus of Fc (fragment of crystallization of IgG) was several-foldmore potent than the TGFβRII-Fc that places the TGFβRII at theN-terminus of Fc (see Example 3). The superb efficacy obtained withanti-PDL1/TGFβ Trap also allays some concerns that the TGFβRII does nottrap TGFβ2. As pointed out by Yang et al., Trends Immunol. 2010;31:220-227, although some tumor types do secrete TGFβ2 initially, as thetumor progresses, the TGFβ in the tumor microenvironment ispredominantly secreted by myeloid-derived suppressor cells, whichsecrete TGFβ1. In addition to showing great promise as an effectiveimmuno-oncology therapeutic, treatment with soluble TGFβRII canpotentially reduce the cardiotoxicity concerns of TGFβ targetingtherapies, especially the TGFβRI kinase inhibitors. This is because ofthe important roles TGFβ2 plays in embryonic development of the heart aswell as in repair of myocardial damage after ischemia and reperfusioninjury (Roberts et al., J Clin Invest. 1992; 90:2056-62).

TGFβ as a Cancer Target

TGFβ had been a somewhat questionable target in cancer immunotherapybecause of its paradoxical roles as the molecular Jekyll and Hyde ofcancer (Bierie et al., Nat Rev Cancer. 2006; 6:506-20). Like some othercytokines, TGFβ activity is developmental stage and context dependent.Indeed TGFβ can act as either a tumor promoter or a tumor suppressor,affecting tumor initiation, progression and metastasis. The mechanismsunderlying this dual role of TGFβ remain unclear (Yang et al., TrendsImmunol. 2010; 31:220-227). Although it has been postulated thatSmad-dependent signaling mediates the growth inhibition of TGFβsignaling, while the Smad independent pathways contribute to itstumor-promoting effect, there are also data showing that theSmad-dependent pathways are involved in tumor progression (Yang et al.,Cancer Res. 2008; 68:9107-11).

Both the TGFβ ligand and the receptor have been studied intensively astherapeutic targets. There are three ligand isoforms, TGFβ1, 2 and 3,all of which exist as homodimers. There are also three TGFβ receptors(TGFβR), which are called TGFβR type I, II and III (López-Casillas etal., J Cell Biol. 1994; 124:557-68). TGFβRI is the signaling chain andcannot bind ligand. TGFβRII binds the ligand TGFβ1 and 3, but not TGFβ2,with high affinity. The TGFβRII/TGFβ complex recruits TGFβRI to form thesignaling complex (Won et al., Cancer Res. 1999; 59:1273-7). TGFβRIII isa positive regulator of TGFβ binding to its signaling receptors andbinds all 3 TGFβ isoforms with high affinity. On the cell surface, theTGFβ/TGFβRIII complex binds TGFβRII and then recruits TGFβRI, whichdisplaces TGFβRIII to form the signaling complex.

Although the three different TGFβ isoforms all signal through the samereceptor, they are known to have differential expression patterns andnon-overlapping functions in vivo. The three different TGF-β isoformknockout mice have distinct phenotypes, indicating numerousnon-compensated functions (Bujak et al., Cardiovasc Res. 2007;74:184-95). While TGFβ1 null mice have hematopoiesis and vasculogenesisdefects and TGFβ3 null mice display pulmonary development and defectivepalatogenesis, TGFβ2 null mice show various developmental abnormalities,the most prominent being multiple cardiac deformities (Bartram et al.,Circulation. 2001; 103:2745-52; Yamagishi et al., Anat Rec. 2012;295:257-67). Furthermore, TGFβ is implicated to play a major role in therepair of myocardial damage after ischemia and reperfusion injury. In anadult heart, cardiomyocytes secrete TGFβ, which acts as an autocrine tomaintain the spontaneous beating rate. Importantly, 70-85% of the TGFβsecreted by cardiomyocytes is TGFβ2 (Roberts et al., J Clin Invest.1992; 90:2056-62). In summary, given the predominant roles of TGFβ1 andTGFβ2 in the tumor microenvironment and cardiac physiology,respectively, a therapeutic agent that neutralizes TGFβ1 but not TGFβ2could provide an optimal therapeutic index by minimizing thecardiotoxicity without compromising the anti-tumor activity. This isconsistent with the findings by the present inventors, who observed alack of toxicity, including cardiotoxicity, for anti-PD-L1/TGFβ Trap inmonkeys.

Therapeutic approaches to neutralize TGFβ include using theextracellular domains of TGFβ receptors as soluble receptor traps andneutralizing antibodies. Of the receptor trap approach, soluble TGFβRIIImay seem the obvious choice since it binds all the three TGFβ ligands.However, TGFβRIII, which occurs naturally as a 280-330 kDglucosaminoglycan (GAG)-glycoprotein, with extracellular domain of 762amino acid residues, is a very complex protein for biotherapeuticdevelopment. The soluble TGFβRIII devoid of GAG could be produced ininsect cells and shown to be a potent TGFβ neutralizing agent(Vilchis-Landeros et al, Biochem J 355:215, 2001). The two separatebinding domains (the endoglin-related and the uromodulin-related) ofTGFβRIII could be independently expressed, but they were shown to haveaffinities 20 to 100 times lower than that of the soluble TGFβRIII, andmuch diminished neutralizing activity (Mendoza et al., Biochemistry.2009; 48:11755-65). On the other hand, the extracellular domain ofTGFβRII is only 136 amino acid residues in length and can be produced asa glycosylated protein of 25-35 kD. The recombinant soluble TGFβRII wasfurther shown to bind TGFβ1 with a K_(D) of 200 pM, which is fairlysimilar to the K_(D) of 50 pM for the full length TGFβRII on cells (Linet al., J Biol Chem. 1995; 270:2747-54). Soluble TGFβRII-Fc was testedas an anti-cancer agent and was shown to inhibit established murinemalignant mesothelioma growth in a tumor model (Suzuki et al., ClinCancer Res. 2004; 10:5907-18). Since TGFβRII does not bind TGFβ2, andTGFβRIII binds TGFβ1 and 3 with lower affinity than TGFβRII, a fusionprotein of the endoglin domain of TGFβRIII and extracellular domain ofTGFβRII was produced in bacteria and was shown to inhibit the signalingof TGFβ1 and 2 in cell based assays more effectively than either TGFβRIIor RIII (Verona et al., Protein Eng Des Sel. 2008; 21:463-73). Despitesome encouraging anti-tumor activities in tumor models, to our knowledgeno TGFβ receptor trap recombinant proteins have been tested in theclinic.

Still another approach to neutralize all three isoforms of the TGFβligands is to screen for a pan-neutralizing anti-TGFβ antibody, or ananti-receptor antibody that blocks the receptor from binding to TGFβ1, 2and 3. GC1008, a human antibody specific for all isoforms of TGFβ, wasin a Phase I/II study in patients with advanced malignant melanoma orrenal cell carcinoma (Morris et al., J Clin Oncol 2008; 26:9028 (Meetingabstract)). Although the treatment was found to be safe and welltolerated, only limited clinical efficacy was observed, and hence it wasdifficult to interpret the importance of anti-TGFβ therapy withoutfurther characterization of the immunological effects (Flavell et al.,Nat Rev Immunol. 2010; 10:554-67). There were also TGFβ-isoform-specificantibodies tested in the clinic. Metelimumab, an antibody specific forTGFβ1 was tested in Phase 2 clinical trial as a treatment to preventexcessive post-operative scarring for glaucoma surgery; andLerdelimumab, an antibody specific for TGFβ2, was found to be safe butineffective at improving scarring after eye surgery in a Phase 3 study(Khaw et al., Ophthalmology 2007; 114:1822-1830). Anti-TGFβRIIantibodies that block the receptor from binding to all three TGFβisoforms, such as the anti-human TGFβRII antibody TR1 and anti-mouseTGFβRII antibody MT1, have also shown some therapeutic efficacy againstprimary tumor growth and metastasis in mouse models (Zhong et al., ClinCancer Res. 2010; 16:1191-205). To date, the vast majority of thestudies on TGFβ targeted anticancer treatment, including small moleculeinhibitors of TGFβ signaling that often are quite toxic, are mostly inthe preclinical stage and the anti-tumor efficacy obtained has beenlimited (Calone et al., Exp Oncol. 2012; 34:9-16; Connolly et al., Int JBiol Sci. 2012; 8:964-78).

The antibody-TGFβ trap of the invention is a bifunctional proteincontaining at least portion of a human TGFβ Receptor II (TGFβRII) thatis capable of binding TGFβ. In one embodiment, the TGFβ trap polypeptideis a soluble portion of the human TGFβ Receptor Type 2 Isoform A (SEQ IDNO: 8) that is capable of binding TGFβ. In a further embodiment, TGFβtrap polypeptide contains at least amino acids 73-184 of SEQ ID NO:8. Inyet a further embodiment, the TGFβ trap polypeptide contains amino acids24-184 of SEQ ID NO:8. In another embodiment, the TGFβ trap polypeptideis a soluble portion of the human TGFβ Receptor Type 2 Isoform B (SEQ IDNO: 9) that is capable of binding TGFβ. In a further embodiment, TGFβtrap polypeptide contains at least amino acids 48-159 of SEQ ID NO:9. Inyet a further embodiment, the TGFβ trap polypeptide contains amino acids24-159 of SEQ ID NO:9. In yet a further embodiment, the TGFβ trappolypeptide contains amino acids 24-105 of SEQ ID NO:9.

Immune Checkpoint Dis-Inhibition

The approach of targeting T cell inhibition checkpoints fordis-inhibition with therapeutic antibodies is an area of intenseinvestigation (for a review, see Pardoll, Nat Rev Cancer. 2012;12:253-264). In one approach, the antibody moiety or antigen bindingfragment thereof targets T cell inhibition checkpoint receptor proteinson the T cell, such as, for example: CTLA-4, PD-1, BTLA, LAG-3, TIM-3,and LAIR1. In another approach, the antibody moiety targets thecounter-receptors on antigen presenting cells and tumor cells (whichco-opt some of these counter-receptors for their own immune evasion),such as, for example: PD-L1 (B7-H1), B7-DC, HVEM, TIM-4, B7-H3, orB7-H4.

The invention contemplates antibody TGFβ traps that target, throughtheir antibody moiety or antigen binding fragment thereof, T cellinhibition checkpoints for dis-inhibition. To that end the presentinventors have tested the anti-tumor efficacy of combining a TGFβ trapwith antibodies targeting various T cell inhibition checkpoint receptorproteins, such as anti-PD-1, anti-PD-L1, anti-TIM-3 and anti-LAG3. Theexperimental results are further detailed in Examples 7-18. The presentinventors found that combining a TGFβ trap with an anti-PD-L1 antibodyexhibited remarkable anti-tumor activity beyond what was observed withthe monotherapies. In contrast, none of the other combinations withantibodies to the targets listed above showed any superior efficacy. Inparticular, one may have expected that a combination treatment of a TGFβtrap with an anti-PD-1 antibody would demonstrate similar activity tothe one observed with anti-PD-L1, as PD-1/PD-L1 are cognate receptorsthat bind to each other to effect the immune checkpoint inhibition.However, this is not what the present inventors have found.

Anti-PD-L1 Antibodies

The invention can include any anti-PD-L1 antibody, or antigen-bindingfragment thereof, described in the art. Anti-PD-L1 antibodies arecommercially available, for example, the 29E2A3 antibody (Biolegend,Cat. No. 329701). Antibodies can be monoclonal, chimeric, humanized, orhuman. Antibody fragments include Fab, F(ab′)2, scFv and Fv fragments,which are described in further detail below.

Exemplary antibodies are described in PCT Publication WO 2013/079174.These antibodies can include a heavy chain variable region polypeptideincluding an HVR-H1, HVR-H2, and HVR-H3 sequence, where:

(a) the HVR-H1 sequence is X₁YX₂MX₃;

(b) the HVR-H2 sequence is SIYPSGGX₄TFYADX₅VKG;

(c) the HVR-H3 sequence is IKLGTVTTVX₆Y;

further where: X₁ is K, R, T, Q, G, A, W, M, I, or S; X₂ is V, R, K, L,M, or I; X₃ is H, T, N, Q, A, V, Y, W, F, or M; X₄ is F or I; X₅ is S orT; X₆ is E or D.

In a one embodiment, X₁ is M, I, or S; X₂ is R, K, L, M, or I; X₃ is For M; X₄ is F or I; X₅ is S or T; X₆ is E or D.

In another embodiment X₁ is M, I, or S; X₂ is L, M, or I; X₃ is F or M;X₄ is I; X₅ is S or T; X₆ is D.

In still another embodiment, X₁ is S; X₂ is I; X₃ is M; X₄ is I; X₅ isT; X₆ is D.

In another aspect, the polypeptide further includes variable regionheavy chain framework sequences juxtaposed between the HVRs according tothe formula:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4).

In yet another aspect, the framework sequences are derived from humanconsensus framework sequences or human germline framework sequences.

In a still further aspect, at least one of the framework sequences isthe following:

HC-FR1 is EVQLLESGGGLVQPGGSLRLSCAASGFTFS;

HC-FR2 is WVRQAPGKGLEWVS;

HC-FR3 is RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR;

HC-FR4 is WGQGTLVTVSS.

In a still further aspect, the heavy chain polypeptide is furthercombined with a variable region light chain including an HVR-L1, HVR-L2,and HVR-L3, where:

(a) the HVR-L1 sequence is TGTX₇X₈DVGX₉YNYVS;

(b) the HVR-L2 sequence is X₁₀VX₁₁X₁₂RPS;

(c) the HVR-L3 sequence is SSX₁₃TX₁₄X₁₅X₁₆X₁₇RV;

further where: X₇ is N or S; X₈ is T, R, or S; X₉ is A or G; X₁₀ is E orD; X₁₁ is I, N or S; X₁₂ is D, H or N; X₁₃ is F or Y; X₁₄ is N or S; X₁₅is R, T or S; X₁₆ is G or S; X₁₇ is I or T.

In another embodiment, X₇ is N or S; X₈ is T, R, or S; X₉ is A or G; X₁₀is E or D; X₁₁ is N or S; X₁₂ is N; X₁₃ is F or Y; X₁₄ is S; X₁₅ is S;X₁₆ is G or S; X₁₇ is T.

In still another embodiment, X₇ is S; X₈ is S; X₉ is G; X₁₀ is D; X₁₁ isS; X₁₂ is N; X₁₃ is Y; X₁₄ is S; X₁₅ is S; X₁₆ is S; X₁₇ is T.

In a still further aspect, the light chain further includes variableregion light chain framework sequences juxtaposed between the HVRsaccording to the formula:(LC-FR1MHVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4).

In a still further aspect, the light chain framework sequences arederived from human consensus framework sequences or human germlineframework sequences.

In a still further aspect, the light chain framework sequences arelambda light chain sequences.

In a still further aspect, at least one of the framework sequence is thefollowing:

LC-FR1 is QSALTQPASVSGSPGQSITISC;

LC-FR2 is WYQQHPGKAPKLMIY;

LC-FR3 is GVSNRFSGSKSGNTASLTISGLQAEDEADYYC;

LC-FR4 is FGTGTKVTVL.

In another embodiment, the invention provides an anti-PD-L1 antibody orantigen binding fragment including a heavy chain and a light chainvariable region sequence, where:

(a) the heavy chain includes an HVR-H1, HVR-H2, and HVR-H3, whereinfurther: (i) the HVR-H1 sequence is X₁YX₂MX³; (ii) the HVR-H2 sequenceis SIYPSGGX₄TFYADX₅VKG; (iii) the HVR-H3 sequence is IKLGTVTTVX₆Y, and;

(b) the light chain includes an HVR-L1, HVR-L2, and HVR-L3, whereinfurther: (iv) the HVR-L1 sequence is TGTX₇X₈DVGX₉YNYVS; (v) the HVR-L2sequence is X₁₀VX₁₁X₁₂RPS; (vi) the HVR-L3 sequence isSSX₁₃TX₁₄X₁₅X₁₆X₁₇RV; wherein: X₁ is K, R, T, Q, G, A, W, M, I, or S; X₂is V, R, K, L, M, or I; X₃ is H, T, N, Q, A, V, Y, W, F, or M; X₄ is For I; X₅ is S or T; X₆ is E or D; X₇ is N or S; X₈ is T, R, or S; X₉ isA or G; X₁₀ is E or D; X₁₁ is I, N, or S; X₁₂ is D, H, or N; X₁₃ is F orY; X₁₄ is N or S; X₁₅ is R, T, or S; X₁₆ is G or S; X₁₇ is I or T.

In one embodiment, X₁ is M, I, or S; X₂ is R, K, L, M, or I; X₃ is F orM; X₄ is F or I; X₅ is S or T; X₆ is E or D; X₇ is N or S; X₈ is T, R,or S; X₉ is A or G; X₁₀ is E or D; X₁₁ is N or S; X₁₂ is N; X₁₃ is F orY; X₁₄ is S; X₁₅ is S; X₁₆ is G or S; X₁₇ is T.

In another embodiment, X₁ is M, I, or S; X₂ is L, M, or I; X₃ is F or M;X₄ is I; X₅ is S or T; X₆ is D; X₇ is N or S; X₈ is T, R, or S; X₉ is Aor G; X₁₀ is E or D; X₁₁ is N or S; X₁₂ is N; X₁₃ is F or Y; X₁₄ is S;X₁₅ is S; X₁₆ is G or S; X₁₇ is T.

In still another embodiment, X₁ is S; X₂ is I; X₃ is M; X₄ is X₅ is T;X₆ is D; X₇ is S; X₈ is S; X₉ is G; X₁₀ is D; X₁₁ is S; X₁₂ is N; X₁₃ isY; X₁₄ is S; X₁₅ is S; X₁₆ is S; X₁₇ is T.

In a further aspect, the heavy chain variable region includes one ormore framework sequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions include one or more framework sequencesjuxtaposed between the HVRs as: (LC-FR1MHVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4).

In a still further aspect, the framework sequences are derived fromhuman consensus framework sequences or human germline sequences.

In a still further aspect, one or more of the heavy chain frameworksequences is the following:

HC-FR1 is EVQLLESGGGLVQPGGSLRLSCAASGFTFS;

HC-FR2 is WVRQAPGKGLEWVS;

HC-FR3 is RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR;

HC-FR4 is WGQGTLVTVSS.

In a still further aspect, the light chain framework sequences arelambda light chain sequences.

In a still further aspect, one or more of the light chain frameworksequences is the following:

LC-FR1 is QSALTQPASVSGSPGQSITISC;

LC-FR2 is WYQQHPGKAPKLMIY;

LC-FR3 is GVSNRFSGSKSGNTASLTISGLQAEDEADYYC;

LC-FR4 is FGTGTKVTVL.

In a still further aspect, the heavy chain variable region polypeptide,antibody, or antibody fragment further includes at least a C_(H)1domain.

In a more specific aspect, the heavy chain variable region polypeptide,antibody, or antibody fragment further includes a C_(H)1, a C_(H)2, anda C_(H)3 domain.

In a still further aspect, the variable region light chain, antibody, orantibody fragment further includes a C_(L) domain.

In a still further aspect, the antibody further includes a C_(H)1, aC_(H)2, a C_(H)3, and a C_(L) domain.

In a still further specific aspect, the antibody further includes ahuman or murine constant region.

In a still further aspect, the human constant region is selected fromthe group consisting of IgG1, IgG2, IgG2, IgG3, IgG4.

In a still further specific aspect, the human or murine constant regionis lgG1.

In yet another embodiment, the invention features an anti-PD-L1 antibodyincluding a heavy chain and a light chain variable region sequence,where:

(a) the heavy chain includes an HVR-H1, an HVR-H2, and an HVR-H3, havingat least 80% overall sequence identity to SYIMM, SIYPSGGITFYADTVKG, andIKLGTVTTVDY, respectively, and

(b) the light chain includes an HVR-L1, an HVR-L2, and an HVR-L3, havingat least 80% overall sequence identity to TGTSSDVGGYNYVS, DVSNRPS, andSSYTSSSTRV, respectively.

In a specific aspect, the sequence identity is 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%.

In yet another embodiment, the invention features an anti-PD-L1 antibodyincluding a heavy chain and a light chain variable region sequence,where:

(a) the heavy chain includes an HVR-H1, an HVR-H2, and an HVR-H3, havingat least 80% overall sequence identity to MYMMM, SIYPSGGITFYADSVKG, andIKLGTVTTVDY, respectively, and

(b) the light chain includes an HVR-L1, an HVR-L2, and an HVR-L3, havingat least 80% overall sequence identity to TGTSSDVGAYNYVS, DVSNRPS, andSSYTSSSTRV, respectively.

In a specific aspect, the sequence identity is 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%.

In a still further aspect, in the antibody or antibody fragmentaccording to the invention, as compared to the sequences of HVR-H1,HVR-H2, and HVR-H3, at least those amino acids remain unchanged that arehighlighted by underlining as follows:

(a) in HVR-H1 SYIMM,

(b) in HVR-H2 SIYPSGGITFYADTVKG,

(c) in HVR-H3 IKLGTVTTVDY;

and further where, as compared to the sequences of HVR-L1, HVR-L2, andHVR-L3 at least those amino acids remain unchanged that are highlightedby underlining as follows:

(a) HVR-L1 TGTSSDVGGYNYVS

(b) HVR-L2 DVSNRPS

(c) HVR-L3 SSYTSSSTRV.

In another aspect, the heavy chain variable region includes one or moreframework sequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions include one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4).

In yet another aspect, the framework sequences are derived from humangermline sequences.

In a still further aspect, one or more of the heavy chain frameworksequences is the following:

HC-FR1 is EVQLLESGGGLVQPGGSLRLSCAASGFTFS;

HC-FR2 is WVRQAPGKGLEWVS;

HC-FR3 is RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR;

HC-FR4 is WGQGTLVTVSS.

In a still further aspect, the light chain framework sequences arederived from a lambda light chain sequence.

In a still further aspect, one or more of the light chain frameworksequences is the following:

LC-FR1 is QSALTQPASVSGSPGQSITISC;

LC-FR2 is WYQQHPGKAPKLMIY;

LC-FR3 is GVSNRFSGSKSGNTASLTISGLQAEDEADYYC;

LC-FR4 is FGTGTKVTVL.

In a still further specific aspect, the antibody further includes ahuman or murine constant region.

In a still further aspect, the human constant region is selected fromthe group consisting of IgG1, IgG2, IgG2, IgG3, IgG4.

In a still further embodiment, the invention features an anti-PD-L1antibody including a heavy chain and a light chain variable regionsequence, where:

(a) the heavy chain sequence has at least 85%sequence identity to the heavy chain sequence:EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMVWRQAPGKGLEWVSSIYPSGGITFYADWKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKL GTVTTVDYWGQGTLVTVSS,and (b) the light chain sequence has at least 85%sequence identity to the light chain sequence:QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRV FGTGTKVTVL.

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

In a still further embodiment, the invention provides for an anti-PD-L1antibody including a heavy chain and a light chain variable regionsequence, where:

(a) the heavy chain sequence has at least 85%sequence identity to the heavy chain sequence:EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYMMMWVRQAPGKGLEVWSSIYPSGGITFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARIK LGTVTTVDYWGQGTLVTVSS,and (b) the light chain sequence has at least 85%sequence identity to the light chain sequence:QSALTQPASVSGSPGQSITISCTGTSSDVGAYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRV FGTGTKVTVL.

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

In another embodiment the antibody binds to human, mouse, or cynomolgusmonkey PD-L1. In a specific aspect the antibody is capable of blockingthe interaction between human, mouse, or cynomolgus monkey PD-L1 and therespective human, mouse, or cynomolgus monkey PD-1 receptors.

In another embodiment, the antibody binds to human PD-L1 with a K_(D) of5×10⁻⁹ M or less, preferably with a K_(D) of 2×10⁻⁹ M or less, and evenmore preferred with a K_(D) of 1×10⁻⁹ M or less.

In yet another embodiment, the invention relates to an anti-PD-L1antibody or antigen binding fragment thereof which binds to a functionalepitope including residues Y56 and D61 of human PD-L1.

In a specific aspect, the functional epitope further includes E58, E60,Q66, R113, and M115 of human PD-L1.

In a more specific aspect, the antibody binds to a conformationalepitope, including residues 54-66 and 112-122 of human PD-L1.

In a further embodiment, the invention is related to an anti-PD-L1antibody, or antigen binding fragment thereof, which cross-competes forbinding to PD-L1 with an antibody according to the invention asdescribed herein.

In a still further embodiment, the invention features proteins andpolypeptides including any of the above described anti-PD-L1 antibodiesin combination with at least one pharmaceutically acceptable carrier.

In a still further embodiment, the invention features an isolatednucleic acid encoding a polypeptide, or light chain or a heavy chainvariable region sequence of an anti-PD-L1 antibody, or antigen bindingfragment thereof, as described herein. In a still further embodiment,the invention provides for an isolated nucleic acid encoding a lightchain or a heavy chain variable region sequence of an anti-PD-L1antibody, wherein:

(a) the heavy chain includes an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 80% sequence identity to SYIMM,SIYPSGGITFYADTVKG, and IKLGTVTTVDY, respectively, or

(b) the light chain includes an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 80% sequence identity to TGTSSDVGGYNYVS,DVSNRPS, and SSYTSSSTRV, respectively.

In a specific aspect, the sequence identity is 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%.

In a further aspect, the nucleic acid sequence for the heavy chain is:

atggagttgc ctgttaggct gttggtgctg atgttctgga ttcctgctag ctccagcgag   60gtgcagctgc tggaatccgg cggaggactg gtgcagcctg gcggctccct gagactgtct  120tgcgccgcct ccggcttcac cttctccagc tacatcatga tgtgggtgcg acaggcccct  180ggcaagggcc tggaatgggt gtcctccatc tacccctccg gcggcatcac cttctacgcc  240gacaccgtga agggccggtt caccatctcc cgggacaact ccaagaacac cctgtacctg  300cagatgaact ccctgcgggc cgaggacacc gccgtgtact actgcgcccg gatcaagctg  360ggcaccgtga ccaccgtgga ctactggggc cagggcaccc tggtgacagt gtcctccgcc  420tccaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc  480acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg  540aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga  600ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac  660atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa  720tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg  780tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag  840gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac  900gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc  960acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 1020tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1080gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcacg ggatgagctg 1140accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1200gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1260gactccgacg gctccttctt cctctatagc aagctcaccg tggacaagag caggtggcag 1320caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1380aagagcctct ccctgtcccc gggtaaa 1407and the nucleic acid sequence for the light chain is:

atggagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cttaagccag  60tccgccctga cccagcctgc ctccgtgtct ggctcccctg gccagtccat caccatcagc 120tgcaccggca cctccagcga cgtgggcggc tacaactacg tgtcctggta tcagcagcac 180cccggcaagg cccccaagct gatgatctac gacgtgtcca accggccctc cggcgtgtcc 240aacagattct ccggctccaa gtccggcaac accgcctccc tgaccatcag cggactgcag 300gcagaggacg aggccgacta ctactgctcc tcctacacct cctccagcac cagagtgttc 360ggcaccggca caaaagtgac cgtgctgggc cagcccaagg ccaacccaac cgtgacactg 420ttccccccat cctccgagga actgcaggcc aacaaggcca ccctggtctg cctgatctca 480gatttctatc caggcgccgt gaccgtggcc tggaaggctg atggctcccc agtgaaggcc 540ggcgtggaaa ccaccaagcc ctccaagcag tccaacaaca aatacgccgc ctcctcctac 600ctgtccctga cccccgagca gtggaagtcc caccggtcct acagctgcca ggtcacacac 660gagggctcca ccgtggaaaa gaccgtcgcc cccaccgagt gctca 705

Further exemplary anti-PD-L1 antibodies that can be used in ananti-PD-L1/TGFβ Trap are described in US patent application publicationUS 2010/0203056. In one embodiment of the invention, the antibody moietyis YW243.55S70. In another embodiment of the invention, the antibodymoiety is MPDL3280A.

In a further embodiment, the invention features an anti-PD-L1 antibodymoiety including a heavy chain and a light chain variable regionsequence, where:

(a) the heavy chain sequence has at least 85%sequence identity to the heavy chain sequence: (SEQ ID NO: 12)EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRH WPGGFDYWGQGTLVTVSS,and (b) the light chain sequence has at least 85%sequence identity to the light chain sequence: (SEQ ID NO: 13)DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQ GTKVEIKR.

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In a further embodiment, the invention features an anti-PD-L1 antibodymoiety including a heavy chain and a light chain variable regionsequence, where:

(a) the heavy chain variable region sequence is: (SEQ ID NO: 12)EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRH WPGGFDYWGQGTLVTVSS,and (b) the light chain variable region sequence is: (SEQ ID NO: 13)DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQ GTKVEIKR.

In a further embodiment, the invention features an anti-PD-L1 antibodymoiety including a heavy chain and a light chain variable regionsequence, where:

(a) the heavy chain variable region sequence is: (SEQ ID NO: 14)EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRH WPGGFDYWGQGTLVTVSA,and (b) the light chain variable region sequence is: (SEQ ID NO: 13)DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQ GTKVEIKR.

Yet further exemplary anti-PD-L1 antibodies that can be used in ananti-PD-L1/TGFβ Trap are described in US patent publication U.S. Pat.No. 7,943,743.

In one embodiment of the invention, the anti-PD-L1 antibody is MDX-1105.

In yet a further embodiment, the anti-PD-L1 antibody is MEDI-4736.

Constant Region

The proteins and peptides of the invention can include a constant regionof an immunoglobulin or a fragment, analog, variant, mutant, orderivative of the constant region. In preferred embodiments, theconstant region is derived from a human immunoglobulin heavy chain, forexample, IgG1, IgG2, IgG3, IgG4, or other classes. In one embodiment,the constant region includes a CH2 domain. In another embodiment, theconstant region includes CH2 and CH3 domains or includes hinge-CH2-CH3.Alternatively, the constant region can include all or a portion of thehinge region, the CH2 domain and/or the CH3 domain.

In one embodiment, the constant region contains a mutation that reducesaffinity for an Fc receptor or reduces Fc effector function. Forexample, the constant region can contain a mutation that eliminates theglycosylation site within the constant region of an IgG heavy chain. Insome embodiments, the constant region contains mutations, deletions, orinsertions at an amino acid position corresponding to Leu234, Leu235,Gly236, Gly237, Asn297, or Pro331 of IgG1 (amino acids are numberedaccording to EU nomenclature). In a particular embodiment, the constantregion contains a mutation at an amino acid position corresponding toAsn297 of IgG1. In alternative embodiments, the constant region containsmutations, deletions, or insertions at an amino acid positioncorresponding to Leu281, Leu282, Gly283, Gly284, Asn344, or Pro378 ofIgG1.

In some embodiments, the constant region contains a CH2 domain derivedfrom a human IgG2 or IgG4 heavy chain. Preferably, the CH2 domaincontains a mutation that eliminates the glycosylation site within theCH2 domain. In one embodiment, the mutation alters the asparagine withinthe Gln-Phe-Asn-Ser (SEQ ID NO: 15) amino acid sequence within the CH2domain of the IgG2 or IgG4 heavy chain. Preferably, the mutation changesthe asparagine to a glutamine. Alternatively, the mutation alters boththe phenylalanine and the asparagine within the Gln-Phe-Asn-Ser (SEQ IDNO: 15) amino acid sequence. In one embodiment, the Gln-Phe-Asn-Ser (SEQID NO: 15) amino acid sequence is replaced with a Gln-Ala-Gln-Ser (SEQID NO: 16) amino acid sequence. The asparagine within theGln-Phe-Asn-Ser (SEQ ID NO: 15) amino acid sequence corresponds toAsn297 of IgG1.

In another embodiment, the constant region includes a CH2 domain and atleast a portion of a hinge region. The hinge region can be derived froman immunoglobulin heavy chain, e.g., IgG1, IgG2, IgG3, IgG4, or otherclasses. Preferably, the hinge region is derived from human IgG1, IgG2,IgG3, IgG4, or other suitable classes. More preferably the hinge regionis derived from a human IgG1 heavy chain. In one embodiment the cysteinein the Pro-Lys-Ser-Cys-Asp-Lys (SEQ ID NO: 17) amino acid sequence ofthe IgG1 hinge region is altered. In a preferred embodiment thePro-Lys-Ser-Cys-Asp-Lys (SEQ ID NO: 17) amino acid sequence is replacedwith a Pro-Lys-Ser-Ser-Asp-Lys (SEQ ID NO: 18) amino acid sequence. Inone embodiment, the constant region includes a CH2 domain derived from afirst antibody isotype and a hinge region derived from a second antibodyisotype. In a specific embodiment, the CH2 domain is derived from ahuman IgG2 or IgG4 heavy chain, while the hinge region is derived froman altered human IgG1 heavy chain.

The alteration of amino acids near the junction of the Fc portion andthe non-Fc portion can dramatically increase the serum half-life of theFc fusion protein (PCT publication WO 01/58957, the disclosure of whichis hereby incorporated by reference). Accordingly, the junction regionof a protein or polypeptide of the present invention can containalterations that, relative to the naturally-occurring sequences of animmunoglobulin heavy chain and erythropoietin, preferably lie withinabout 10 amino acids of the junction point. These amino acid changes cancause an increase in hydrophobicity. In one embodiment, the constantregion is derived from an IgG sequence in which the C-terminal lysineresidue is replaced. Preferably, the C-terminal lysine of an IgGsequence is replaced with a non-lysine amino acid, such as alanine orleucine, to further increase serum half-life. In another embodiment, theconstant region is derived from an IgG sequence in which theLeu-Ser-Leu-Ser (SEQ ID NO: 19) amino acid sequence near the C-terminusof the constant region is altered to eliminate potential junctionalT-cell epitopes. For example, in one embodiment, the Leu-Ser-Leu-Seramino acid sequence is replaced with an Ala-Thr-Ala-Thr (SEQ ID NO: 20)amino acid sequence. In other embodiments, the amino acids within theLeu-Ser-Leu-Ser (SEQ ID NO: 19) segment are replaced with other aminoacids such as glycine or proline. Detailed methods of generating aminoacid substitutions of the Leu-Ser-Leu-Ser (SEQ ID NO: 19) segment nearthe C-terminus of an IgG1, IgG2, IgG3, IgG4, or other immunoglobulinclass molecule have been described in U.S. Patent Publication No.2003/0166877, the disclosure of which is hereby incorporated byreference.

Suitable hinge regions for the present invention can be derived fromIgG1, IgG2, IgG3, IgG4, and other immunoglobulin classes. The IgG1 hingeregion has three cysteines, two of which are involved in disulfide bondsbetween the two heavy chains of the immunoglobulin. These same cysteinespermit efficient and consistent disulfide bonding formation between Fcportions. Therefore, a preferred hinge region of the present inventionis derived from IgG1, more preferably from human IgG1. In someembodiments, the first cysteine within the human IgG1 hinge region ismutated to another amino acid, preferably serine. The IgG2 isotype hingeregion has four disulfide bonds that tend to promote oligomerization andpossibly incorrect disulfide bonding during secretion in recombinantsystems. A suitable hinge region can be derived from an IgG2 hinge; thefirst two cysteines are each preferably mutated to another amino acid.The hinge region of IgG4 is known to form interchain disulfide bondsinefficiently. However, a suitable hinge region for the presentinvention can be derived from the IgG4 hinge region, preferablycontaining a mutation that enhances correct formation of disulfide bondsbetween heavy chain-derived moieties (Angal S, et al. (1993) Mol.Immunol., 30:105-8).

In accordance with the present invention, the constant region cancontain CH2 and/or CH3 domains and a hinge region that are derived fromdifferent antibody isotypes, i.e., a hybrid constant region. Forexample, in one embodiment, the constant region contains CH2 and/or CH3domains derived from IgG2 or IgG4 and a mutant hinge region derived fromIgG1. Alternatively, a mutant hinge region from another IgG subclass isused in a hybrid constant region. For example, a mutant form of the IgG4hinge that allows efficient disulfide bonding between the two heavychains can be used. A mutant hinge can also be derived from an IgG2hinge in which the first two cysteines are each mutated to another aminoacid. Assembly of such hybrid constant regions has been described inU.S. Patent Publication No. 2003/0044423, the disclosure of which ishereby incorporated by reference.

In accordance with the present invention, the constant region cancontain one or more mutations described herein. The combinations ofmutations in the Fc portion can have additive or synergistic effects onthe prolonged serum half-life and increased in vivo potency of thebifunctional molecule. Thus, in one exemplary embodiment, the constantregion can contain (i) a region derived from an IgG sequence in whichthe Leu-Ser-Leu-Ser (SEQ ID NO: 19) amino acid sequence is replaced withan Ala-Thr-Ala-Thr (SEQ ID NO: 20) amino acid sequence; (ii) aC-terminal alanine residue instead of lysine; (iii) a CH2 domain and ahinge region that are derived from different antibody isotypes, forexample, an IgG2 CH2 domain and an altered IgG1 hinge region; and (iv) amutation that eliminates the glycosylation site within the IgG2-derivedCH2 domain, for example, a Gln-Ala-Gln-Ser (SEQ ID NO: 16) amino acidsequence instead of the Gln-Phe-Asn-Ser (SEQ ID NO: 15) amino acidsequence within the IgG2-derived CH2 domain.

Antibody Fragments

The proteins and polypeptides of the invention can also includeantigen-binding fragments of antibodies. Exemplary antibody fragmentsinclude scFv, Fv, Fab, F(ab′)₂, and single domain VHH fragments such asthose of camelid origin.

Single-chain antibody fragments, also known as single-chain antibodies(scFvs), are recombinant polypeptides which typically bind antigens orreceptors; these fragments contain at least one fragment of an antibodyvariable heavy-chain amino acid sequence (V_(H)) tethered to at leastone fragment of an antibody variable light-chain sequence (V_(L)) withor without one or more interconnecting linkers. Such a linker may be ashort, flexible peptide selected to assure that the properthree-dimensional folding of the V_(L) and V_(H) domains occurs oncethey are linked so as to maintain the target moleculebinding-specificity of the whole antibody from which the single-chainantibody fragment is derived. Generally, the carboxyl terminus of theV_(L) or V_(H) sequence is covalently linked by such a peptide linker tothe amino acid terminus of a complementary V_(L) and V_(H) sequence.Single-chain antibody fragments can be generated by molecular cloning,antibody phage display library or similar techniques. These proteins canbe produced either in eukaryotic cells or prokaryotic cells, includingbacteria.

Single-chain antibody fragments contain amino acid sequences having atleast one of the variable regions or CDRs of the whole antibodiesdescribed in this specification, but are lacking some or all of theconstant domains of those antibodies. These constant domains are notnecessary for antigen binding, but constitute a major portion of thestructure of whole antibodies. Single-chain antibody fragments maytherefore overcome some of the problems associated with the use ofantibodies containing part or all of a constant domain. For example,single-chain antibody fragments tend to be free of undesiredinteractions between biological molecules and the heavy-chain constantregion, or other unwanted biological activity. Additionally,single-chain antibody fragments are considerably smaller than wholeantibodies and may therefore have greater capillary permeability thanwhole antibodies, allowing single-chain antibody fragments to localizeand bind to target antigen-binding sites more efficiently. Also,antibody fragments can be produced on a relatively large scale inprokaryotic cells, thus facilitating their production. Furthermore, therelatively small size of single-chain antibody fragments makes them lesslikely than whole antibodies to provoke an immune response in arecipient.

Fragments of antibodies that have the same or comparable bindingcharacteristics to those of the whole antibody may also be present. Suchfragments may contain one or both Fab fragments or the F(ab′)₂ fragment.The antibody fragments may contain all six CDRs of the whole antibody,although fragments containing fewer than all of such regions, such asthree, four or five CDRs, are also functional.

Protein Production

The antibody-cytokine trap proteins are generally producedrecombinantly, using mammalian cells containing a nucleic acidengineered to express the protein. Although one example of a suitablecell line and protein production method is described in Examples 1 and2, a wide variety of suitable vectors, cell lines and protein productionmethods have been used to produce antibody-based biopharmaceuticals andcould be used in the synthesis of these antibody-cytokine trap proteins.

Therapeutic Indications

The anti-PD-L1/TGFβ Trap proteins described in the application can beused to treat cancer or reduce tumor growth in a patient. Exemplarycancers include colorectal, breast, ovarian, pancreatic, gastric,prostate, renal, cervical, myeloma, lymphoma, leukemia, thyroid,endometrial, uterine, bladder, neuroendocrine, head and neck, liver,nasopharyngeal, testicular, small cell lung cancer, non-small cell lungcancer, melanoma, basal cell skin cancer, squamous cell skin cancer,dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma,glioma, sarcoma, mesothelioma, and myelodisplastic syndromes.

The cancer or tumor to be treated with an anti-PD-L1/TGFβ Trap may beselected based on the expression or elevated expression of PD-L1 andTGFβ in the tumor, the correlation of their expression levels withprognosis or disease progression, and preclinical and clinicalexperience on the sensitivity of the tumor to treatments targeting PD-L1and TGFβ. Such cancers or tumors include but are not limited tocolorectal, breast, ovarian, pancreatic, gastric, prostate, renal,cervical, bladder, head and neck, liver, non-small cell lung cancer,melanoma, Merkel cell carcinoma, and mesothelioma.

Pharmaceutical Compositions

The present invention also features pharmaceutical compositions thatcontain a therapeutically effective amount of a protein describedherein. The composition can be formulated for use in a variety of drugdelivery systems. One or more physiologically acceptable excipients orcarriers can also be included in the composition for proper formulation.Suitable formulations for use in the present invention are found inRemington's Pharmaceutical Sciences, Mack Publishing Company,Philadelphia, Pa., 17th ed., 1985. For a brief review of methods fordrug delivery, see, e.g., Langer (Science 249:1527-1533, 1990).

The pharmaceutical compositions are intended for parenteral, intranasal,topical, oral, or local administration, such as by a transdermal means,for therapeutic treatment. The pharmaceutical compositions can beadministered parenterally (e.g., by intravenous, intramuscular, orsubcutaneous injection), or by oral ingestion, or by topical applicationor intraarticular injection at areas affected by the vascular or cancercondition. Additional routes of administration include intravascular,intra-arterial, intratumor, intraperitoneal, intraventricular,intraepidural, as well as nasal, ophthalmic, intrascleral, intraorbital,rectal, topical, or aerosol inhalation administration. Thus, theinvention provides compositions for parenteral administration thatcomprise the above mention agents dissolved or suspended in anacceptable carrier, preferably an aqueous carrier, e.g., water, bufferedwater, saline, PBS, and the like. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents, detergents and thelike. The invention also provides compositions for oral delivery, whichmay contain inert ingredients such as binders or fillers for theformulation of a tablet, a capsule, and the like. Furthermore, thisinvention provides compositions for local administration, which maycontain inert ingredients such as solvents or emulsifiers for theformulation of a cream, an ointment, and the like.

These compositions may be sterilized by conventional sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as-is, or lyophilized, the lyophilizedpreparation being combined with a sterile aqueous carrier prior toadministration. The pH of the preparations typically will be between 3and 11, more preferably between 5 and 9 or between 6 and 8, and mostpreferably between 7 and 8, such as 7 to 7.5. The resulting compositionsin solid form may be packaged in multiple single dose units, eachcontaining a fixed amount of the above-mentioned agent or agents, suchas in a sealed package of tablets or capsules. The composition in solidform can also be packaged in a container for a flexible quantity, suchas in a squeezable tube designed for a topically applicable cream orointment.

The optimal dose of the antibody-TGFβ trap is based on the percentreceptor occupancy by the antibody moiety to achieve maximal therapeuticeffect because the cytokine trap is used in a large excess. For example,the therapeutic dose for a monoclonal antibody targeting a cellularreceptor is determined such that the trough level is around 10 to 100μg/ml, i.e., 60 to 600 nM (for antibody with a dissociation constant(K_(D)) of 6 nM, this trough level would ensure that between 90 to 99%of the target receptors on the cells are occupied by the antibody). Thisis in large excess of cytokines, which are typically present in pg tong/ml in circulation.

The optimal dose of antibody-TGFβ trap polypeptide of the invention willdepend on the disease being treated, the severity of the disease, andthe existence of side effects. The optimal dose can be determined byroutine experimentation. For parenteral administration a dose between0.1 mg/kg and 100 mg/kg, alternatively between 0.5 mg/kg and 50 mg/kg,alternatively, between 1 mg/kg and 25 mg/kg, alternatively between 2mg/kg and 10 mg/kg, alternatively between 5 mg/kg and 10 mg/kg isadministered and may be given, for example, once weekly, once everyother week, once every third week, or once monthly per treatment cycle.

EXAMPLES

The invention now being generally described, will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the scope of theinvention in any way.

Example 1—DNA Construction and Protein Expression

Anti-PD-L1/TGFβ Trap is an anti-PD-L1 antibody-TGFβ Receptor II fusionprotein. The light chain of the molecule is identical to the light chainof the anti-PD-L1 antibody (SEQ ID NO:1). The heavy chain of themolecule (SEQ ID NO:3) is a fusion protein comprising the heavy chain ofthe anti-PD-L1 antibody (SEQ ID NO:2) genetically fused to via aflexible (Gly₄Ser)₄Gly linker (SEQ ID NO:11) to the N-terminus of thesoluble TGFβ Receptor II (SEQ ID NO:10). At the fusion junction, theC-terminal lysine residue of the antibody heavy chain was mutated toalanine to reduce proteolytic cleavage. For expression ofanti-PD-L1/TGFβ Trap, the DNA encoding the anti-PD-L1 light chain (SEQID NO:4) and the DNA encoding the anti-PD-L1/TGFβ Receptor II (SEQ IDNO:5) in either the same expression vector or separate expressionvectors were used to transfect mammalian cells using standard protocolsfor transient or stable transfection. Conditioned culture media wereharvested and the anti-PD-L1/TGFβ Trap fusion protein was purified bystandard Protein A Sepharose chromatography. The purified proteincomprising one anti-PD-L1 antibody and two soluble TGFβ Receptor IImolecules (FIG. 1A) has an estimated molecular weight (MW) of about 190kilodaltons on size exclusion chromatography and SDS-polyacrylamideelectrophoresis under non-reducing conditions. Under reducingconditions, the light and heavy chains have apparent MW of 28 and 75kilodaltons, respectively (FIG. 1B).

The anti-PD-L1(mut)/TGFβ Trap fusion protein, which contains ananalogous heavy chain fusion polypeptide (SEQ ID NO:7) and a light chainwith the mutations A31G, D52E, R99Y in the variable region that abrogatethe binding to PD-L1 (SEQ ID NO:6), was similarly prepared. It was usedin subsequent experiments as a TGFβ Trap control.

Example 2—Production of Anti-PD-L1/TGFβ Trap as a Biotherapeutic

The anti-PD-L1/TGFβ Trap produced by transient transfection of humanembryonic kidney 293 (HEK) cells was found to contain varying degrees ofa clipped species, which appeared as a faint band with an apparent MW ofabout 60 kD on SDS-PAGE under reducing conditions (FIG. 1B). This bandwas confirmed to be the heavy chain of the anti-PD-L1/TGFβ Trap cleavedat a site in the N-terminal portion of TGFβRII close to the fusionjunction.

Stable clones expressing anti-PD-L1/TGFβ Trap were generated in theCHO-S host cell line, which was pre-adapted for growth in serum-freemedia in suspension culture. Cells were transfected with an expressionvector containing a gene encoding the anti-PD-L1-TGFβRII protein and aglutamine synthetase selection marker. Subsequent selection of stableintegrants was made with L-methionine sulfoximine (MSX). Anti-PD-L1/TGFβTrap expressing cell lines were generated using a minipool approach,followed by the deposition of single cells into 384-well plates, using aBeckton-Dickinson fluorescence activated cell sorter (FACS Aria II).Growth, productivity, and protein quality were evaluated in a genericplatform fed-batch assay. Based on these analyses, 14 clones wereselected as lead candidates for further studies. A stability study withthe clones was carried out to ˜90 PDL (population doubling level) fromresearch cell banks established during scale up of clones. At theconclusion of minipool development it was discovered that the heavychain-linker-TGFβRII subunit underwent clipping, as was seen intransient expression. All clones in the stability study produced theclipped species, although it was shown in the protein A-purifiedmaterial that the percent clipped species relative to the intact subunitvaried with each clone. In addition, an improved purification processconsisting a protein A chromatography followed by strong cation exchangewas developed to reduce co-purification of the clipped species. Evenwith the improved process, purified material with the required finallevels of clipped species of <5% could only be achieved using clonesproducing low levels of clipping. Based on these combined analyses,clone 02B15 was selected as the final candidate clone. Analysis ofanti-PD-L1/TGFβ Trap expressed by this clone at zero PDL, thirty PDL,sixty PDL and ninety PDL shows that the percentage of clipping did notincrease with population doubling levels (FIG. 2).

Example 3—Fluorescence-Activated Cell Sorting (FACS) Analysis of Bindingof Anti-PD-L1/TGFβ Trap and Controls to Human PD-L1 on Cells

The binding of anti-PD-L1 antibody and fusion proteins on HEK cellsstably transfected to express human PD-L1 was studied using thefollowing procedure.

The following exemplary procedure was used determine PD-L1 binding byFACS:

-   -   a. 50 μl serial dilutions of test samples were set up in FACS        buffer.    -   b. 50 μl of HEK cells stably transfected to express human PD-L1        at 5×10⁶ cells/ml were dispensed to the wells with test samples        and mixed.    -   c. Plate(s) were incubated in the dark on ice for 1 hour.    -   d. Cells were pelleted at 300×g for 5 minutes.    -   e. Supernatant was decanted and cells were resuspended in 300 μl        FACS buffer and re-pelleted at 300×g for 5.    -   f. Sample rinse was repeated.    -   g. Cells were resuspended in 100 μl FACS buffer containing        DyLight 488 conjugated whole IgG Goat Anti-Human IgG, Fcγ (1:300        diluted).    -   h. Plate(s) was incubated in the dark on ice for 45 minutes.    -   i. Cells were pelleted at 300×g for 5.    -   j. Supernatant was decanted and cells were resuspended in 300 μl        FACS buffer and re-pelleted at 300×g for 5 minutes    -   k. Sample rinse was repeated and cells were finally resuspended        in 200 μl FACS buffer.    -   l. Data was acquired on FACS Caliber and was analyzed using        Microsoft Excel. EC50 was calculated using non-linear regression        (Sigmoidal dose-response) with Graphpad Prism 5.

As shown in FIG. 3, FACS analysis showed that the anti-PD-L1/TGFβ Trapfusion protein retains similar binding affinity as the positive controlanti-PD-L1 antibody on HEK cells stably transfected to express humanPD-L1 (HEK/PD-L1 cells). The EC50's for anti-PD-L1/TGFβ Trap andanti-PD-L1 are 0.116 μg/ml (0.64 nM) and 0.061 μg/ml (0.41 nM),respectively. The observed MFI (mean fluorescent intensity) was specificto binding to human PD-L1 since no MFI was observed on the parental HEKcells that were not transfected. The anti-PD-L1(mut)/TGFβ Trap negativecontrol did not show any binding to the HEK cells stably transfected toexpress human PD-L1.

Example 4—Determination of Ability of Anti-PD-L1/TGFβ Trap to InhibitTGFβ Induced Phosphorylation of SMAD3

The ability of anti-PD-L1/TGFβ Trap to neutralize TGFβ was determinedusing 4T1 cells carrying a SMAD3-luciferase reporter. In the assaydetailed below, inhibition of TGFβ-induced phosphorylation of SMAD3 wasmeasured using a luciferase reporter under the control of the SMAD3promoter.

An exemplary assay to evaluate potency to inhibit TGFβ-induced reporteractivity was performed as follows.

-   -   1. One day prior to the study, 4T1 cells carrying        SMAD3-luciferase reporter were fed.    -   2. On day 0, cells were plated in a Biocoat 96-well plate at a        concentration of 5×10⁴ cells/well in 100 μl of fresh media and        incubated overnight at 37° C. and 5% CO₂.    -   3. On day 1:        -   i. 50 μl of fresh complete media containing indicated            concentration of anti-PD-L1/TGFβ trap samples to be tested            or its controls was added to the wells and incubated for one            hour. All samples were tested in triplicates.        -   ii. 50 μl of fresh complete media containing 20 ng/ml human            TGFβ was added to each well and samples were incubated            overnight (final concentration in the well is 5 ng/ml).    -   4. On day 2:        -   i. 100 μl culture supernatant was removed and 100 μl fresh            complete media, containing 150 μg/ml D-Luciferin was added,            and samples were incubated for at least five minutes.        -   ii. Luminescence was measured using Envision 2104 plate            reader by recording CPM.    -   5. Data was analyzed using MS Excel or Graphpad prism 5.        Luciferase activity was recorded as CPM. Inhibitory Activity of        (%) was calculated using the following equation:

Inhibition (%)=(1−CPM of sample/CPM max of anti-PD-L1 treatedsample)×100

-   -   6. Nonlinear regression fit was carried out using Sigmoidal        dose-response (variable slope) of Graphpad prism 5. IC50 values        were calculated.

FIG. 4 shows that anti-PD-L1/TGFβ Trap inhibits TGFβ-induced pSMAD3reporter activity in a dose dependent manner. The fact that theanti-PD-L1(mut)/TGFβ Trap control had comparable potency and IC50(concentration required to inhibit 50% of the maximal activity) plus thefact that the anti-PD-L1 antibody had no effect showed that thisinhibition of signaling is independent of anti-PD-L1 activity.Surprisingly, anti-PD-L1/TGFβ Trap was several-fold more potent thanTGFβRII-Fc (R&D Systems), which places the TGFβRII at the N-terminusinstead of the C-terminus of the fusion protein. It is also noteworthythat anti-PD-L1/TGFβ Trap is significantly more potent than 1D11(GC1008), the anti-TGFβ antibody that was tested in patients withadvanced malignant melanoma or renal cell carcinoma (Morris et al., JClin Oncol 2008; 26:9028 (Meeting abstract)). In this assay, 1D11 andTGFβRII-Fc showed similar activity.

Example 5—Pharmacokinetic (PK) Analysis in Mice

Eighteen male C57BL/6 mice, 5-6 weeks old, were randomly assigned to 3groups (N=6/group), and each group received one of the three proteins(anti-PD-L1/TGFβ Trap, anti-PD-L1(mut)/TGFβ Trap, and anti-PD-L1). Mousebody weight was recorded before dosing. After a brief warm-up under aheating lamp, each mouse received 120 μg of protein in 200 μlintravenously (IV) via the tail vein regardless of its body weight. Eachgroup dosed with the same protein was further divided into 2 subgroups(n=3). Blood samples were alternately taken from each of two subgroups,i.e. one subgroup was withdrawn for blood samples at 1 h, 24 h, 72 h,and 168 h, whereas another subgroup was for blood samples at 7 h, 48 h,120 h, and 240 h. At each time point, approximate 50 μl of blood sampleswere collected from each mouse via tail vein using a heparinized microglass capillary (100 μl in capacity). The blood sample was thentransferred to a tube pre-coated with Li-Heparin and kept at 4° C.Within 10 min of collection, the blood samples were spun at 14,000 rpmfor 10 min. At least 20 μl of plasma sample was transferred into a newset of pre-labeled tubes and stored at −20° C. until the day ofanalysis.

The ELISA to measure total human IgG used goat anti-Human IgG (H+L)(heavy and light chains) (Jackson ImmunoResearch Laboratories) coatedwells for capture and peroxidase-AffiniPure mouse anti-Human IgG,F(ab′)2 (Jackson ImmunoResearch Laboratories) for detection. The ELISAto measure fully functional anti-PD-L1 antibody and/or fusion proteinused PD-L1-Fc (extracellular domain of human PD-L1 fused to Fc) coatedwells (coated at 1.25 μg/ml) for capture and peroxidase-AffiniPure mouseanti-Human IgG, F(ab′)2 for detection. The ELISA to measure fullyfunctional anti-PD-L1 and intact TGFβRII used PD-L1-Fc coated wells forcapture and biotinylated anti-human TGFβRII (R&D Systems) for detection.

FIG. 5A shows that the anti-PD-L1/TGFβ Trap fusion protein had a PKprofile very similar to that of the anti-PD-L1 antibody. For example, asmeasured by the total human IgG ELISA, the serum concentrations at the168 hr time point of anti-PD-L1/TGFβ Trap and anti-PD-L1 were 16.8 and16.2 μg/ml, respectively, and the respective area under the curve (AUC)from 0 to 168 hr were 4102 and 3841 hr-μg/ml. Similarly, when the serumconcentrations were measured by the total functional anti-PD-L1 ELISA,the serum concentrations at the 168 hr time point of anti-PD-L1/TGFβTrap and anti-PD-L1 were 9.5 and 11.1 μg/ml, respectively, and therespective AUC from 0 to 168 hr were 3562 and 3086 hr-μg/ml. The serumconcentration of intact anti-PD-L1/TGFβ Trap fusion protein wasdetermined by the ELISA, which detects fully functional anti-PD-L1 andthe fused TGFβRII. In this case, the serum concentration ofanti-PD-L1/TGFβ Trap was 5.9 μg/ml at the 168 hr time point and the AUC(0 to 168 hr) was 2656 hr-ng/ml, which were somewhat lower than thosefrom the fully functional anti-PD-L1 ELISA, presumably due todegradation of the TGFβRII moiety after receptor-mediated endocytosis.Antibody binding to PD-L1 has been shown to result in PD-L1-mediatedendocytosis, and an antibody-X fusion protein is known to undergodegradation of the X moiety after receptor-mediated endocytosis (Gillieset al., Clin Cancer Res. 2002; 8:210-6). This is supported by thefinding in FIG. 5 that when the antibody moiety does not bind PD-L1, asin the anti-PD-L1(mut)/TGFβ Trap control, the exposure is about 3 timeshigher, with a serum concentration of 53 μg/ml at the 168 hr time pointand AUC (0 to 168 hr) of 9585 hr-μg/ml, suggesting that at least part ofthe clearance is receptor-mediated.

In order to confirm the 3-fold difference in exposure betweenanti-PD-L1/TGFβ Trap and anti-PD-L1(mut)/TGFβ Trap, the pharmacokineticsexperiment was repeated and the concentrations of the intact fusionproteins in the serum samples were determined. Mice (B6.129S2 femalemice, 8 wks old, Jackson Lab) were injected with anti-PD-L1/TGFβ Trap oranti-PD-L1(mut)/TGFβ Trap (164 μg/mouse). The serum concentrations ofthe two fusion proteins were measured by an ELISA using anti-human IgGFab (Jackson Immunoresearch, West Grove, Pa.) for capture andbiotinylated anti-human TGFβRII (R&D Systems, Minneapolis, Minn.) andperoxidase-conjugated streptavidin (Zymed/ThermoFisher Scientific, GrandIsland, N.Y.) to detect intact anti-PD-L1/TGFβ Trap proteins. The serumconcentrations of the intact fusion proteins at various time points wereshown in the Table below and plotted in FIG. 5B. The total area underthe curve (AUC) up to 336 hr is 11781 hr-μg/ml for anti-PD-L1/TGFβ Trapand 35575 hr-μg/ml for anti-PD-L1(mut)/TGFβ Trap (Table 1), thereforeconfirming the three-fold higher exposure of the Trap control molecule.

TABLE 1 Exposures of anti-PD-L1/TGFβ Trap and the anti-PD-L1(mut)/TGFβTrap control as determined by the area under the curve (AUC) in thepharmacokinetics graph in FIG. 5B. AUC (h * μg/ml) Time (h)Anti-PD-L1/TGFβ Trap Anti-PD-L1(mut)/TGFβ Trap 7 72 173 24 1161 2789 481306 3511 72 1113 2968 120 2327 5192 168 2014 5225 240 2159 7530 3361629 8188 total 11781 35575

Example 6—PD-L1 Target-Mediated Endocytosis of Anti-PD-L1/TGFβ Trap

Receptor-mediated endocytosis was studied using the Alexa Fluor 488quenching techniques according to manufacturer's protocol (LifeTechnologies, Carlsbad, Calif.). Briefly, HEK cells expressing PD-L1(HEK/PD-L1 cells) were incubated with 10 μg/ml Alexa Fluor488-conjugated anti-PD-L1/TGFβ Trap on ice for about 1 hr and washed 4times with cold media. Washed cells were then pulsed at 37° C. for 0.25,0.5, 0.75, 1, 1.5, 2, 3 and 4 hr to allow internalization. Cell samplesat each time point were then divided into two portions. One portion wasincubated on ice and total fluorescence from the Alexa Fluor488-conjugated anti-PD-L1/TGFβ Trap bound on the cell surface andinternalized was measured; the other portion was incubated withanti-Alexa Fluor 488 at 4° C. for about an hour and the non-quenchablefluorescence from the internalized Alexa Fluor 488-conjugatedanti-PD-L1/TGFβ Trap was measured. A graph showing a time course of thenon-quenchable and total mean fluorescence intensity (MFI) ofanti-PD-L1/TGFβ Trap at 37° C. is shown in FIG. 6A. Thereceptor-mediated internalization kinetics is very similar to that ofthe anti-PD-L1 antibody, which is shown in FIG. 6B. The percentage ofreceptor-mediated internalization of anti-PD-L1/TGFβ Trap and anti-PD-L1on HEK/PD-L1 cells at various time points at 37° C. is shown in FIG. 6C,using the following formula to account for the fact that the quenchingby the anti-Alexa Fluor 488 is not 100%:

Internalized fluorescence=Total MFI−(Total MFI−Non-quenchableMFI)/Quenching efficiency

Example 7—Anti-PD-L1/TGFβ Trap Demonstrated a Superior Anti-Tumor Effectthat is Synergistic of Anti-PD-L1 and TGFβ Trap Activities in the EMT-6(Breast Carcinoma) Subcutaneous Model

8-12 week old female Jh (Igh-J^(tm1Dhu)) Balb/C mice (Taconic Farms,Hudson, N.Y.) were inoculated with 0.5×10⁶ viable EMT6 cells in 0.1 mlPBS on the right flanks subcutaneously. About five days later, whentumors reached an average size of 20-30 mm³, mice were sorted intogroups (N=10) so that the average tumor sizes of all groups weresimilar, and treatment by intravenous injections was initiated (Day 0).Group 1 received 400 μg of isotype antibody control three times weekly(or “eod” (every other day); Group 2 received 400 μg of anti-PD-L1antibody three times weekly; Group 3 received 164 μg ofanti-PD-L1(mut)/TGFβ Trap three times weekly; Group 4 received 492 μg ofanti-PD-L1/TGFβ Trap three times weekly; Group 5 received 492 μg ofanti-PD-L1/TGFβ Trap twice weekly (equimolar to 400 μg of anti-PD-L1antibody); Group 6 received 164 μg of anti-PD-L1/TGFβ Trap three timesweekly; and Group 7 received 55 μg of anti-PD-L1/TGFβ Trap three timesweekly. Body weights were measured twice weekly to monitor toxicity.Tumor volumes were determined at different time points using theformula: tumor volume (mm³)=length×width×height×0.5236. Any mice withtumors over 2500 mm³ were sacrificed following the institute's animalhealth protocol. Anti-tumor efficacy was reported as a T/C ratio, whereT and C are the average tumor volumes of the group treated with antibodyor fusion protein, and the group treated with the isotype control,respectively.

All the treatments were well tolerated. The inhibition of tumor growthby the various treatments is shown in FIG. 7A, which showed the averagetumor volumes of the surviving mice, and FIG. 7B, which showed theindividual tumor volume of the surviving mice, noting that mice withtumors over 2500 mm³ had to be euthanized. Anti-PD-L1/TGFβ Trapdemonstrated potent anti-tumor efficacy, achieving T/C ratios of 0.30,0.40, and 0.44 for the high (492 μg, Group 4), medium (164 μg, Group 6),and low (55 μg, Group 7) dose groups, respectively on Day 28). While theanti-PD-L1 antibody (Group 2, T/C=0.87, p>0.05, on Day 16, the last dayfor which the average tumor volume of all the mice were available, i.e.,before mice with tumors over 2500 mm³ were euthanized) or the TGFβ Trapcontrol (Group 2, T/C=0.97 on Day 16, p>0.05) alone had marginalefficacy in this model, combining the two agents in a single moleculeresulted in profound synergistic anti-tumor effect. This is evident inthe median survival times observed for the 492 μg dose (58 and greaterthan 80 days, respectively, for three times weekly dosing and twiceweekly dosing) and 164 μg dose (35 days) of the fusion protein (log ranktest: p<0.0001) (FIG. 7C). Importantly, anti-PD-L1/TGFβ Trap at themedium dose of 164 μg (Group 6), with a median survival of 35 days, wasfar more efficacious than the same dose of anti-PD-L1(mut)/TGFβ Trap(Group 3) or three times the equivalent dose of anti-PD-L1 (Group 2),both of which yielded a median survival of 22 days, respectively (logrank test: p<0.0001). This synergistic anti-tumor activity is especiallystriking because the exposure of the TGFβ Trap moiety of the 164 μg doseof PD-L1(mut)/TGFβ Trap should be about 3 times higher than that of the164 μg dose of PD-L1/TGFβ Trap due to receptor-mediated clearance of thelatter (see Examples 5 and 6). It is remarkable that tumors in micewhich received the high dose of anti-PD-L1/TGFβ Trap continued toregress after dosing was stopped on Day 18 (3 of 10 from Group 4 and 6of 10 from Group 5 with complete regressions at day 78), demonstratingthe long-lasting immunologic anti-tumor effect of targeting the twoimmunosuppressive mechanisms simultaneously (FIG. 7C). It is alsonoteworthy that the efficacy for Group 4 is not any better than that ofGroup 5, suggesting that the dose of 492 μg administered twice weeklywas near the saturating dose, or was a more optimal dosing regimen thanthe 492 μg administered three times weekly.

The protective effect of the anti-tumor immunity elicited by theanti-PD-L1/TGFβ Trap treatment was evident when the mice with tumors incomplete regression were challenged with 25,000 viable EMT6 cellsinjected subcutaneously. While all ten naïve mice in a control groupdeveloped tumors to an average tumor volume of 726 mm³ by Day 18 postchallenge, none of the eleven mice previously treated with PD-L1/TGFβTrap (three from Group 4, six from Group 5, and one each from Groups 6and 7) showed any sign of tumor growth.

Example 8—Anti-PD-L1/TGF-β Trap Showed Profound Synergistic Anti-TumorActivity in the MC38 (Colorectal Carcinoma) Subcutaneous Tumor Model

8-12 week old female B6.129S2-Ighm^(tm1Cgn)/J mice (Jackson Laboratory,Bar Harbor, Me.) were injected with 0.5×10⁶ viable MC38 tumor cells in0.1 ml PBS subcutaneously into the right flank. About eight days later,when average tumor size reached about 80-100 mm³, mice were sorted intogroups (N=10) so that the average tumor sizes of all groups weresimilar, and treatment by intravenous injections was initiated (Day 0).Group 1 received 400 μg of isotype antibody control; Group 2 received400 μg of anti-PD-L1 antibody; Group 3 received 133 μg of anti-PD-L1antibody; Group 4 received 492 μg of anti-PD-L1(mut)/TGFβ Trap; Group 5received 164 μg of anti-PD-L1(mut)/TGFβ Trap; Group 6 received 492 μg ofanti-PD-L1/TGFβ Trap; and Group 7 received 164 μg of anti-PD-L1/TGFβTrap. The treatment was administered three times weekly for two weeks.Body weights were measured twice weekly to monitor toxicity. Tumorvolumes were determined at different time points using the formula:tumor volume (mm³)=length×width×height×0.5236. Any mice with tumors over2500 mm³ were sacrificed following the institute's animal healthprotocol. Anti-tumor efficacy was reported as a T/C ratio, where T and Care the average tumor volumes of the group treated with antibody orfusion protein, and the group treated with the isotype control,respectively.

All the treatments were well tolerated. The inhibition of tumor growthby the various treatments is shown in FIG. 8. On day 19 of the study,anti-PD-L1/TGFβ Trap demonstrated potent dose-dependent anti-tumorefficacy, achieving T/C ratios of 0.18 (p<0.001) and 0.38 (p<0.001) forthe high (492 μg, Group 6) and low (164 μg, Group 7) dose groups,respectively. On the other hand, neither anti-PD-L1 oranti-PD-L1(mut)/TGFβ Trap showed any anti-tumor activity at all.Therefore, a profound syngergistic anti-tumor activity was obtained whenthe anti-PD-L1 antibody and the TGFβ Trap moiety were combined into onemolecule to target these two immunosuppressive mechanismssimultaneously.

Example 9—Anti-PDL1/TGFβ Trap was Effective in the EMT-6 OrthotopicModel of Metastatic Breast Cancer

8-12 week old female Jh (Igh-J^(tm1Dhu)) Balb/C mice (Taconic Farms,Hudson, N.Y.) were inoculated with 0.25×10⁶ viable EMT6 cells in 0.1 mlPBS into the right mammary pad. About a week later, when average tumorsize reached about 50 mm³, mice were sorted into groups (N=10) so thatthe average tumor sizes of all groups were similar, and treatment byintravenous injections was initiated (Day 0). Group 1 received 133 μg ofisotype antibody control; Group 2 received 133 μg of anti-PD-L1antibody; Group 3 received 164 μg of anti-PD-L1 (mut)/TGFβ Trap; Group 4received 164 μg of anti-PD-L1/TGFβ Trap; and Group 5 received acombination of 133 μg of anti-PD-L1 and 164 μg of anti-PD-L1(mut)/TGFβTrap. Treatment was repeated on Days 0, 2, 4, 7, 9, 11 (i.e. 3 timesweekly for two weeks). Body weights were measured twice weekly tomonitor toxicity. Tumor volumes were determined at different time pointsusing the formula: tumor volume (mm³)=length×width×height×0.5236. Anymice with tumors over 2500 mm³ were sacrificed following the institute'sanimal health protocol. Anti-tumor efficacy was reported as a T/C ratio,where T and C are the average tumor volumes of the group treated withantibody or fusion protein, and the group treated with the isotypecontrol, respectively.

All treatments were well tolerated. The inhibition of tumor growth bythe various treatments is shown in FIG. 9. Anti-PD-L1/TGFβ Trapdemonstrated potent anti-tumor efficacy, achieving T/C ratio of 0.03(p<0.001) on Day 21. On the other hand, equimolar doses of anti-PD-L1 oranti-PD-L1(mut)/TGFβ Trap were less efficacious, giving T/C ratios of0.31 (p<0.001 vs. Group 1; p<0.001 vs. Group 4) and 0.68 (p<0.001 vs.Group 1; p<0.001 vs. Group 4), respectively. The combination therapy ofequimolar doses of anti-PD-L1 and anti-PD-L1(mut)/TGFβ Trap achievedalmost identical anti-tumor efficacy as the fusion protein, although theexposure of the TGFβ Trap of the fusion protein (Group 4) was estimatedto be about 3-fold lower than that of the anti-PD-L1(mut)/TGFβ Trap inthe combination (Group 5) based on pharmacokinetics analysis (seeExample 5). It is also remarkable that the tumors in Groups 4 and 5continued to regress after the last day of dosing, e.g., average tumorsize decreased from 212 mm³ on Day 11, the last day of dosing, to 26 mm³on Day 24 for anti-PD-L1/TGFβ Trap treatment, demonstrating thelong-lasting immunologic anti-tumor effect of targeting the twoimmunosuppressive mechanisms simultaneously.

Example 10—Anti-PD-L1/TGFβ Trap has Better Anti-Tumor Efficacy than theCombination of Anti-PD-L1 and TGFβ Trap in an Intramuscular MC38Colorectal Carcinoma Model

8-12 week old female B6.129S2-Ighm^(tm1Cgn)/J mice (Jackson Laboratory,Bar Harbor, Me.) were injected with 0.5×10⁶ viable MC38 tumor cells in0.1 ml PBS intramuscularly in the right thigh. About a week later, whenaverage tumor size reaches about 50 mm³, mice were sorted into groups(N=8) so that the average tumor sizes of all groups were similar, andtreatment by intravenous injections was initiated (Day 0) and repeatedagain two days later (Day 2). Group 1 received 400 μg of isotypeantibody control; Group 2 received 400 μg of anti-PD-L1 antibody; Group3 received 133 μg of anti-PD-L1 antibody; Group 4 received 164 μg ofanti-PD-L1(mut)/TGFβ Trap; Group 5 received 492 μg of anti-PD-L1/TGFβTrap; Group 6 received 164 μg of anti-PD-L1/TGFβ Trap; and Group 7received a combination of 133 μg of anti-PD-L1 and 164 μg ofanti-PD-L1(mut)/TGFβ Trap. Body weights were measured twice weekly tomonitor toxicity. Tumor volumes were determined at different time pointsusing the formula: tumor volume (mm³)=length×width×height×0.5236. Anymice with tumors over 2500 mm³ were sacrificed following the institute'sanimal health protocol. Anti-tumor efficacy was reported as a T/C ratio,where T and C are the average tumor volumes of the group treated withantibody or fusion protein, and the group treated with the isotypecontrol, respectively.

All the treatments were well tolerated. The inhibition of tumor growthby the various treatments is shown in FIG. 10. Anti-PD-L1/TGFβ Trapdemonstrated very potent anti-tumor efficacy, achieving T/C ratios of0.024 (p<0.001) and 0.052 (p<0.001) for the high (492 μg, Group 5) andlow (164 μg, Group 6) dose groups, respectively, on Day 15. On the otherhand, equimolar doses of anti-PD-L1 were less efficacious, giving T/Cratios of 0.59 (p<0.001) and 0.45 (p<0.001) for the high (400 μg, Group2) and low (133 μg, Group 3) dose groups, respectively.Anti-PD-L1(mut)/TGFβ Trap at 164 μg (Group 4) was completelyineffective, and it should be pointed out that although this dose isequimolar with the low dose anti-PD-L1/TGFβ Trap group (Group 6), theexposure of the TGFβ Trap should be fairly similar to that of the highdose anti-PD-L1/TGFβ Trap group (Group 5) because of the differences inpharmacokinetics (see Example 5). Therefore, the data demonstrated thatanti-PD-L1/TGFβ Trap had potent synergistic anti-tumor activity in thismodel. It is especially noteworthy that, anti-PD-L1/TGFβ Trap was moreefficacious than the combination therapy of equimolar doses ofanti-PD-L1 and anti-PD-L1(mut)/TGFβ Trap, which had a T/C ratio of 0.16(p<0.001 vs. Group 1 and p>0.05 vs. Group 6) despite a higher TGFβ Trapexposure of about threefold (see Example 5). In addition,anti-PD-L1/TGFβ Trap treatment resulted in 4 out of 10 mice withcomplete tumor regression, while the combination of anti-PD-L1 and theTrap control induced complete regression in only 2 out of 10 mice (datanot shown). It is also remarkable that the tumors in the mice treatedwith anti-PD-L1/TGFβ Trap continued to regress after the last day ofdosing on day 2, and stayed completely regressed thereafter (until atleast Day 102), demonstrating the profound and long-lasting immunologicanti-tumor effect of this fusion protein. Without being bound by theory,the data supports a mechanism in which the anti-PD-L1/TGFβ Trap fusionprotein not only exploits the synergistic effect of blocking the twomajor immune escape pathways, but is superior to the combination therapydue to the targeting of the tumor microenvironment by a single molecularentity. Many immunosuppressive cytokines secreted by tumor cells orsubverted immune cells (e.g. tumor associated macrophages,myeloid-derived suppressor cells) have autocrines or paracrinefunctions. Therefore, anti-PD-L1/TGFβ Trap has the capability to deliverthe TGFβ Trap to the tumor microenvironment via binding to PD-L1+ tumorcells, where the Trap neutralizes the locally secreted TGFβ. Inaddition, instead of acting just like a sink for bound TGFβ thataccumulates in circulation, anti-PD-L1/TGFβ Trap bound TGFβ could beeffectively destroyed through the PD-L1 receptor-mediated endocytosis(Examples 5 and 6).

Example 11—Treatment with Anti-PDL1/TGFβ Trap or the Combination ofAnti-PD-L1 and TGFβ Trap Control at Equivalent Exposure in the EMT-6Orthotopic Model of Metastatic Breast Cancer

At equimolar doses, anti-PDL1/TGFβ Trap had similar efficacy as thecombination of anti-PD-L1 and TGFβ Trap control in the orthotopic EMT-6breast cancer model (Example 9). In the following study the efficacy ofanti-PDL1/TGFβ Trap or the combination of anti-PD-L1 and TGFβ Trapcontrol administered for equivalent exposure was tested.

8-12 week old female Jh (Igh-J^(tm1Dhu)) Balb/C mice (Taconic Farms,Hudson, N.Y.) were inoculated with 0.25×10⁶ viable EMT6 cells in 0.1 mlPBS into the right mammary pad. About a week later, when average tumorsize reached about 80 mm³, mice were sorted into groups (N=12) so thatthe average tumor sizes of all groups were similar, and treatment byintravenous injections was initiated on Day 0 and repeated 7 days later.Group 1 received 133 μg of isotype antibody control; Group 2 received164 μg of anti-PD-L1/TGFβ Trap; Group 3 received 55 μg ofanti-PD-L1/TGFβ Trap; Group 4 received a combination of 133 μg ofanti-PD-L1 and 55 μg of anti-PD-L1(mut)/TGFβ Trap; and Group 5 receiveda combination of 44.3 μg of anti-PD-L1 and 18.3 μg ofanti-PD-L1(mut)/TGFβ Trap. Body weights were measured twice weekly tomonitor toxicity. Tumor volumes were determined at different time pointsusing the formula: tumor volume (mm³)=length×width×height×0.5236. Anymice with tumors over 2500 mm³ were sacrificed following the institute'sanimal health protocol. Anti-tumor efficacy is reported as a T/C ratio,where T and C are the average tumor volumes of the group treated withantibody or fusion protein, and the group treated with the isotypecontrol, respectively.

All the treatments were well tolerated. Anti-PD-L1/TGFβ Trap and thecombination therapy demonstrated potent anti-tumor efficacy at both doselevels tested.

Example 12—Anti-PD-L1/TGF-β Trap has Better Antitumor Efficacy than theCombination of Anti-PD-L1 and TGFβ Trap Administered to Give EquivalentExposure in an Intramuscular MC38 Colorectal Carcinoma Model

The results in Example 10 suggested that at equimolar doses theanti-PD-L1/TGF-β Trap has better antitumor efficacy than the combinationof anti-PD-L1 and TGFβ Trap control even though the in vivo exposure ofanti-PD-L1(mut)/TGFβ Trap control is about 3 times that ofanti-PD-L1/TGFβ Trap (Example 5). In a follow-up study the anti-tumorefficacy of anti-PD-L1/TGFβ Trap and the combination of anti-PD-L1 andanti-PD-L1(mut)/TGFβ Trap based on equal exposure was compared. Lowerdoses than in Example 10 were administered to avoid dosing nearsaturating levels.

8-12 week old female B6.129S2-Ighm^(tm1Cgn)/J mice (Jackson Laboratory,Bar Harbor, Me.) were injected with 0.5×10⁶ viable MC38 tumor cells in0.1 ml PBS intramuscularly in the right thigh. A week later, whenaverage tumor size reached about 200 mm³, mice were sorted into groups(N=12) so that the average tumor sizes of all groups were similar.Treatment by intravenous injections was initiated (Day 0) and repeatedagain on Day 4. Group 1 received 133 μg of isotype antibody control;Group 2 received 164 μg of anti-PD-L1/TGFβ Trap; Group 3 received 55 μgof anti-PD-L1/TGFβ Trap; Group 4 received a combination of 133 μg ofanti-PD-L1 and 55 μg of anti-PD-L1(mut)/TGFβ Trap; and Group 5 receiveda combination of 44.3 μg of anti-PD-L1 and 18.3 μg ofanti-PD-L1(mut)/TGFβ Trap. Body weights were measured twice weekly tomonitor toxicity. Tumor volumes were determined at different time pointsusing the formula: tumor volume (mm³)=length×width×height×0.5236. Anymice with tumors over 2500 mm³ were sacrificed following the institute'sanimal health protocol. Anti-tumor efficacy is reported as a T/C ratio,where T and C are the average tumor volumes of the group treated withantibody or fusion protein, and the group treated with the isotypecontrol, respectively.

All the treatments were well tolerated. Anti-PD-L1/TGFβ Trapdemonstrated very potent anti-tumor efficacy, achieving T/C ratios of0.13 (p<0.001) and 0.19 (p<0.001) for the intermediate (164 μg, Group 2,called intermediate dose relative to the high dose of 492 μg that seemedto be saturating in Example 10) and low (55 μg, Group 3) dose groups,respectively, on Day 9. On the other hand, the combination of anti-PD-L1and anti-PD-L1(mut)/TGFβ Trap were less efficacious, giving T/C ratiosof 0.34 (p<0.001) and 0.37 (p<0.001) for the intermediate (Group 4) andlow (Group 5) dose groups, respectively (FIG. 12A or Table). It isespecially noteworthy that when administered to give equivalent in vivoexposure of the anti-PD-L1 antibody and the TGFβ Trap component,anti-PD-L1/TGFβ Trap was significantly more efficacious than thecombination therapy of anti-PD-L1 and anti-PD-L1(mut)/TGFβ Trap at bothdose levels (at the intermediate dose, T/C of 0.13 for anti-PD-L1/TGFβTrap vs. 0.34 for the combination p<0.0001 (FIG. 12B); at the low dose,T/C of 0.19 for anti-PD-L1/TGFβ Trap vs. 0.37 for the combinationp<0.0001 (FIG. 12C)).

Example 13—Anti-PD-L1(YW)/TGFβ Trap has Superior Anti-Tumor Effect thatis Synergistic of Anti-PD-L1 and TGFβ Trap Activities in the EMT-6(Breast Carcinoma) Orthotopic Model

YW243.55S70 is a human antibody that recognizes both human and murinePD-L1 (US Patent Application Publication No. US2010/0203056 A1). Itsvariable region sequence of the heavy chain (VH) and variable regionsequence of the light chain (VL) (provided as SEQ ID NO: 14 and SEQ IDNO: 13, respectively) were used to replace the corresponding variableregion sequences of the anti-PD-L1/TGFβ Trap described in Example 1 togive anti-PD-L1(YW)/TGFβ Trap by standard molecular biology techniques.After construction of the DNA coding for anti-PD-L1(YW)/TGFβ Trap, theantibody fusion protein was expressed as described in Example 1. Theanti-PD-L1 antibody YW243.55S70 is similarly expressed for comparison ofefficacy in murine tumor models.

8-12 week old female Jh (Igh-Jtm1Dhu) Balb/C mice (Taconic Farms,Hudson, N.Y.) were inoculated with 0.25×10⁶ viable EMT6 cells in 0.1 mlPBS into the right mammary pad. About a week later, when average tumorsize reached about 50-100 mm³, mice were sorted into groups (N=10) sothat the average tumor sizes of all groups were similar, and treatmentby intravenous injections was initiated (Day 0). Group 1 received 133 μgof isotype antibody control; Group 2 received 133 μg of anti-PD-L1(YW)antibody; Group 3 received 164 μg of anti-PD-L1(mut)/TGFβ Trap; Group 4received 164 μg of anti-PD-L1(YW)/TGFβ Trap; and Group 5 received acombination of 133 μg of anti-PD-L1(YW) and 164 μg of anti-PD-L1(mut)/TGFβ Trap. Treatment was repeated on Days 4 and 7. Body weightswere measured twice weekly to monitor toxicity. Tumor volumes weredetermined at different time points using the formula tumor volume(mm³)=length×width×height×0.5236. Any mice with tumors over 2500 mm³were sacrificed following the institute's animal health protocol.Anti-tumor efficacy is reported as a T/C ratio, where T and C are theaverage tumor volumes of the group treated with antibody or fusionprotein, and the group treated with the isotype control, respectively.

All the treatments were well tolerated. The inhibition of tumor growthby the various treatments is shown in FIG. 13A, which showed the averagetumor volumes of the mice on Day 17, the last day for which the averagetumor volume of all the mice were available, i.e., before mice withtumors over 2500 mm³ were euthanized. Anti-PD-L1(YW)/TGFβ Trapdemonstrated potent anti-tumor efficacy, achieving a T/C ratio of 0.25(p<0.0001) that is slightly better than that of the combinationtreatment in Group 5 (T/C=0.31, p<0.0001), but superior to that of theanti-PD-L1(YW) antibody in Group 2 (T/C=0.57, p<0.0001) and the TGFβTrap control in Group 3 (T/C=0.66, p<0.0001). The synergistic anti-tumoreffect of the antibody fusion protein also resulted in prolongedsurvival of the treated mice, as shown in FIG. 13B. The anti-PD-L1/TGFβTrap treated group had a median survival time of 65 days, which wassignificantly better than that of the anti-PD-L1(YW) antibody treatedgroup (24 days) or the TGFβ Trap control treated group (21 days). Italso compares favorably with the median survival time of 53.5 days forthe combination treatment group. Despite dosing stopped after day 7, thecontinual tumor growth inhibition and the prolonged survival of theanti-PD-L1(YW)/TGFβ Trap treated mice demonstrate the long-lastingimmunologic anti-tumor effect resulting from dual blockade of the twomajor immunosuppressive pathways.

Example 14—Anti-PD-L1(YW)/TGF-β Trap has Superior Anti-Tumor Effect thatis Synergistic of Anti-PD-L1 and TGFβ Trap Activities in the MC38(Colorectal Carcinoma) Intramuscular Tumor Model

8-12 week old female B6.129S2-Ighm^(tm1Cgn)/J mice (Jackson Laboratory,Bar Harbor, Me.) were injected with 0.5×10⁶ viable MC38 tumor cells in0.1 ml PBS intramuscularly in the right thigh. About a week later, whenaverage tumor size reaches about 150-200 mm³, mice were sorted intogroups (N=10) so that the average tumor sizes of all groups weresimilar, and treatment by intravenous injections was initiated (Day 0)and repeated again four days later (Day 4). Group 1 received 133 μg ofisotype antibody control; Group 2 received 133 μg of anti-PD-L1(YW)antibody; Group 3 received 164 μg of anti-PD-L1(mut)/TGFβ Trap; Group 4received 164 μg of anti-PD-L1(YW)/TGFβ Trap; and Group 5 received acombination of 133 μg of anti-PD-L1(YW) and 164 μg ofanti-PD-L1(mut)/TGFβ Trap. Body weights were measured twice weekly tomonitor toxicity. Tumor volumes were determined at different time pointsusing the formula tumor volume (mm³)=length×width×height×0.5236. Anymice with tumors over 2500 mm³ were sacrificed following the institute'sanimal health protocol. Anti-tumor efficacy was reported as a T/C ratio,where T and C are the average tumor volumes of the group treated withantibody or fusion protein, and the group treated with the isotypecontrol, respectively.

All the treatments were well tolerated. The inhibition of tumor growthby the various treatments is shown in FIG. 14A, which showed the averagetumor volumes of the mice on Day 10, the last day for which the averagetumor volume of all the mice were available. Anti-PD-L1(YW)/TGFβ Trapdemonstrated very potent anti-tumor efficacy, achieving a T/C ratio of0.14 (p<0.0001) that is slightly better than that of the combinationtreatment in Group 5 (T/C=0.19, p<0.0001), but superior to that of theanti-PD-L1(YW) antibody in Group 2 (T/C=0.34, p<0.0001) and the TGFβTrap control in Group 3 (T/C=0.99, p<0.0001), which had no activity inthis model. The anti-tumor efficacy of anti-PD-L1(YW)/TGFβ Trap wasfurther confirmed by tumor weight measurements taken on Day 11. By thistime, the isotype control group had to be euthanized because the tumorshad grown beyond 2500 mm³. Therefore, the experiment was terminated andall the groups were euthanized and the tumor weights determined. Theindividual tumor weights are shown in FIG. 14B. The analysis of tumorweights confirmed that anti-PD-L1(YW)/TGFβ Trap therapy significantlyinhibited MC38 tumor growth (T/C=0.13; p<0.0001). The efficacy ofanti-PD-L1(YW)/TGFβ Trap was significantly better than that observedwith anti-PD-L1 (T/C=0.37; p=0.003) or the TGFβ Trap control (T/C=1.0,p<0.0001). The anti-tumor efficacy of anti-PD-L1(YW)/TGFβ Trap, based onthe tumor weight analysis, was not statistically better than the micetreated with the combination of anti-PD-L1 and the TGFβ Trap control(T/C=0.17; p=0.96).

Example 15—Combination Treatment of Anti-PD-1 and TGFβ Trap do notProvide any Additive Anti-Tumor Effect in an EMT-6 (Breast Carcinoma)Orthotopic Model

In this study we tested if the combination treatment of anti-PD-1 andTGFβ Trap provides any additive anti-tumor effect in the EMT-6orthotopic model. CT-011, also known as pidiluzumab, is a humanizedanti-human PD1 antibody that was tested in the clinic for treatment ofhematological malignancies (Berger et al, Clin Cancer Res. 2008;14:3044-3051). It also recognizes murine PD-1 and has shown anti-tumoractivity that synergizes with cyclophosphamide and vaccine treatment insyngeneic tumor models (Mkrtichyan et al., Eur J Immunol. 2011;41:2977-86). The VH and VL sequences of CT-011 were used to produce arecombinant antibody with human IgG1/kappa constant regions by standardmolecular biology techniques.

8-12 week old female Jh (Igh-JtmlDhu) Balb/C mice (Taconic Farms,Hudson, N.Y.) were inoculated with 0.25×10⁶ viable EMT6 cells in 0.1 mlPBS into the right mammary pad. About a week later, when average tumorsize reached about 100 mm³, mice were sorted into groups (N=10) so thatthe average tumor sizes of all groups were similar, and treatment byintravenous injections was initiated (Day 0). Group 1 received 364 μg ofisotype antibody control; Group 2 received 164 μg ofanti-PD-L1(mut)/TGFβ Trap, which served as the TGFβ Trap control; Group3 received 200 μg of anti-PD-1(CT-011); and Group 4 received acombination of 200 μg of anti-PD-1(CT-011) and 164 μg ofanti-PD-L1(mut)/TGFβ Trap control. Treatment was repeated on Days 2, 4,7, 9, and 11, i.e. 3 times weekly for two weeks. Body weights weremeasured twice weekly to monitor toxicity. Tumor volumes were determinedat different time points using the formula tumor volume(mm³)=length×width×height×0.5236. Any mice with tumors over 2500 mm³were sacrificed following the institute's animal health protocol.Anti-tumor efficacy was reported as a T/C ratio, where T and C are theaverage tumor volumes of the group treated with antibody or fusionprotein, and the group treated with the isotype control, respectively.

All the treatments were well tolerated. Anti-PD-1(CT-011) showed verymodest anti-tumor efficacy (T/C=0.87, p>0.05) in this model, while itscombination with the TGFβ Trap control had the same efficacy as the TGFβTrap control alone (FIG. 15).

Example 16—Combination Treatment of Anti-PD-1 and TGFβ Trap do notProvide any Additive Anti-Tumor Effect in an MC38 (Colorectal Carcinoma)Intramuscular Tumor Model

In this study we tested if the combination treatment of anti-PD-1 andTGFβ Trap provides any additive anti-tumor effect in the intramuscularMC38 colorectal tumor model. 8-12 week old femaleB6.129S2-Ighm^(th1Cgn)/J mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected with 0.5×10⁶ viable MC38 tumor cells in 0.1 mL PBSintramuscularly in the right thigh. About a week later, when averagetumor size reaches about 190 mm³, mice are sorted into groups (N=10) sothat the average tumor sizes of all groups are similar, and treatment byintravenous injections is initiated (Day 0). Group 1 received 364 μg ofisotype antibody control on Days 0, 2, 4, and 7; Group 2 received 164 μgof the anti-PD-L1(mut)/TGFβ Trap control on Days 0 and 2; Group 3received 200 μg of anti-PD-1(CT-011) on Days 0, 2, 4, and 7; and Group 4received a combination of 200 μg of anti-PD-1(CT-011) on Days 0, 2, 4,and 7, and 164 μg of anti-PD-L1(mut)/TGFβ Trap control on Days 0 and 2.Body weights were measured twice weekly to monitor toxicity. Tumorvolumes were determined at different time points using the formula tumorvolume (mm³)=length×width×height×0.5236. Any mice with tumors over 2500mm³ were sacrificed following the institute's animal health protocol.Anti-tumor efficacy was reported as a T/C ratio, where T and C are theaverage tumor volumes of the group treated with antibody or fusionprotein, and the group treated with the isotype control, respectively.

All the treatments were well tolerated. Anti-PD-1(CT-011) showed verymodest anti-tumor efficacy (T/C=0.87, p>0.05), while theanti-PD-L1(mut)/TGFβ Trap control had no efficacy in this model, as seenin previous examples. The combination of anti-PD-1(CT-011) with the TGFβTrap control had no efficacy at all (FIG. 15).

Example 17—Combination Treatment of TGFβ Trap with Either Anti-LAG3 orAnti-TIM-3 do not Provide any Additive Anti-Tumor Effect in an EMT-6(Breast Carcinoma) Orthotopic Model

In this study we tested if the combination treatment of TGFβ Trap witheither anti-LAG3 or anti-TIM3 provides any additive anti-tumor effect inthe orthotopic EMT-6 breast tumor model. The anti-LAG3 antibody used isa rat IgG1 monoclonal anti-murine LAG3 antibody C9B7W (BioXcell,Beverly, Mass.), which was shown to synergize with anti-murine PD-1treatment in syngeneic tumor models (Woo et al, Cancer Res, 2011;72:917-27). The anti-TIM-3 antibody used is a rat IgG2a monoclonalanti-murine TIM3 antibody RMT3-23 (BioXcell, Beverly, Mass.), which alsowas shown to synergize with anti-murine PD-1 treatment in syngeneictumor models, although its efficacy as a single agent was relativelymodest (Ngiow et al, Cancer Res, 2011; 71:3540-51).

8-12 week old female Jh (Igh-Jtm1Dhu) Balb/C mice (Taconic Farms,Hudson, N.Y.) were inoculated with 0.25×10⁶ viable EMT6 cells in 0.1 mlPBS into the right mammary pad. About a week later, when average tumorsize reached about 110 mm³, mice were sorted into groups (N=9) so thatthe average tumor sizes of all groups were similar, and treatment byintravenous injections was initiated (Day 0). Group 1 received 133 μg ofisotype antibody control; Group 2 received 164 μg of theanti-PD-L1(mut)/TGFβ Trap control; Group 3 received 200 μg of anti-LAG3;Group 4 received 250 μg of anti-TIM3; Group 5 received a combination of200 μg of anti-LAG3 and 164 μg of anti-PD-L1(mut)/TGFβ Trap control; andGroup 6 received a combination of 250 μg of anti-TIM3 and 164 μg ofanti-PD-L1(mut)/TGFβ Trap control. Treatment was repeated on Days 2, 4,7, 9, and 11, i.e. 3 times weekly for two weeks. Body weights weremeasured twice weekly to monitor toxicity. Tumor volumes were determinedat different time points using the formula tumor volume(mm³)=length×width×height×0.5236. Any mice with tumors over 2500 mm³were sacrificed following the institute's animal health protocol.Anti-tumor efficacy was reported as a T/C ratio, where T and C are theaverage tumor volumes of the group treated with antibody or fusionprotein, and the group treated with the isotype control, respectively.

As observed previously, the anti-PD-L1(mut)/TGFβ Trap control (Group 2)showed very modest efficacy in this EMT-6 model. Anti-TIM3 (Group 4) asa single agent showed a similarly modest efficacy as the Trap control,and in combination therapy with the Trap control (Group 6) showed noadditive effect. Anti-LAG3 either as a single agent (Group 3) or incombination therapy with the Trap control (Group 5) did not show anyefficacy.

Example 18—Combination Treatment of TGFβ Trap with Either Anti-LAG3 orAnti-TIM-3 do not Provide any Additive Anti-Tumor Effect in an MC38(Colorectal Carcinoma) Intramuscular Tumor Model

In this study we tested if the combination treatment of TGFβ Trap witheither anti-LAG3 (C9B7W) or anti-TIM3 (RMT3-23) provides any additiveanti-tumor effect in the intramuscular MC38 colorectal tumor model.

8-12 week old female B6.129S2-Ighm^(tm1Cgn)/J mice (Jackson Laboratory,Bar Harbor, Me.) were injected with 0.5×10⁶ viable MC38 tumor cells in0.1 mL PBS intramuscularly in the right thigh. About a week later, whenaverage tumor size reaches about 50 mm³, mice were sorted into groups(N=8) so that the average tumor sizes of all groups were similar, andtreatment by intravenous injections is initiated (Day 0). Group 1received 133 μg of isotype antibody control; Group 2 received 164 μg ofthe anti-PD-L1(mut)/TGFβ Trap control; Group 3 received 200 μg ofanti-LAG3; Group 4 received 250 μg of anti-TIM3; Group 5 received acombination of 200 μg of anti-LAG3 and 164 μg of anti-PD-L1(mut)/TGFβTrap control; and Group 6 received a combination of 250 μg of anti-TIM3and 164 μg of anti-PD-L1(mut)/TGFβ Trap control. Treatment was repeatedon Days 2, 4, 7, 9, 11, 15 and 18. Body weights were measured twiceweekly to monitor toxicity. Tumor volumes were determined at differenttime points using the formula tumor volume(mm³)=length×width×height×0.5236. Any mice with tumors over 2500 mm³were sacrificed following the institute's animal health protocol.Anti-tumor efficacy was reported as a T/C ratio, where T and C are theaverage tumor volumes of the group treated with antibody or fusionprotein, and the group treated with the isotype control, respectively.

As observed previously, the anti-PD-L1(mut)/TGFβ Trap control (Group 2)did not have any efficacy in this MC38 model. Anti-LAG3 as a singleagent (Group 3) showed a moderate efficacy, achieving a T/C of 0.66(p<0.05). However, combination with the Trap control (Group 5) did notimprove its efficacy. Anti-TIM3 either as a single agent (Group 4) or incombination therapy with the Trap control (Group 6) did not show anyefficacy.

SEQUENCES SEQ ID NO: 1Peptide sequence of the secreted anti-PD-L1 lambda light chainQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVEGTGTKVTVLGQPKANPTVTLEPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVT HEGSTVEKTVAPTECSSEQ ID NO: 2 Peptide sequence of the secreted H chain of anti- PDL1EVQLLESGGGLVQPGGSLRLSCAASGFTESSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFELYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 3Peptide sequence of the secreted H chain of anti- PDL1/TGFβ TrapEVQLLESGGGLVQPGGSLRLSCAASGFTESSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFELYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGAGGGGSGGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE YNTSNPD SEQ ID NO: 4DNA sequence from the translation initiation codonto the translation stop codon of the anti-PD-L1lambda light chain (the leader sequence precedingthe VL is the signal peptide from urokinase plasminogen activator)atgagggccctgctggctagactgctgctgtgcgtgctggtcgtgtccgacagcaagggcCAGTCCGCCCTGACCCAGCCTGCCTCCGTGTCTGGCTCCCCTGGCCAGTCCATCACCATCAGCTGCACCGGCACCTCCAGCGACGTGGGCGGCTACAACTACGTGTCCTGGTATCAGCAGCACCCCGGCAAGGCCCCCAAGCTGATGATCTACGACGTGTCCAACCGGCCCTCCGGCGTGTCCAACAGATTCTCCGGCTCCAAGTCCGGCAACACCGCCTCCCTGACCATCAGCGGACTGCAGGCAGAGGACGAGGCCGACTACTACTGCTCCTCCTACACCTCCTCCAGCACCAGAGTGTTCGGCACCGGCACAAAAGTGACCGTGCTGggccagcccaaggccaacccaaccgtgacactgttccccccatcctccgaggaactgcaggccaacaaggccaccctggtctgcctgatctcagatttctatccaggcgccgtgaccgtggcctggaaggctgatggctccccagtgaaggccggcgtggaaaccaccaagccctccaagcagtccaacaacaaatacgccgcctcctcctacctgtccctgacccccgagcagtggaagtcccaccggtcctacagctgccaggtcacacacgagggctccaccgtggaaaagaccgtcgcccccaccg agtgctcaTGASEQ ID NO: 5 DNA sequence from the translation initiation codonto the translation stop codon (mVK SP leader:small underlined; VH: capitals; IgG1m3 with K to Amutation: small letters; (G4S)x4-G linker: boldcapital letters; TGFβRII: bold underlined smallletters; two stop codons: bold underlined capital letters)atggaaacagacaccctgctgctgtgggtgctgctgctgtgggtgcccggctccacaggcGAGGTGCAGCTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCAGCTACATCATGATGTGGGTGCGACAGGCCCCTGGCAAGGGCCTGGAATGGGTGTCCTCCATCTACCCCTCCGGCGGCATCACCTTCTACGCCGACACCGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCCGGATCAAGCTGGGCACCGTGACCACCGTGGACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCCgctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtgctGGCGGCGGAGGAAGCGGAGGAGGTGGCAGCGGTGGCGGTGGCTCCGGCGGAGGTGGCTCCGGA atccctccccacgtgcagaagtccgtgaacaacgacatgatcgtgaccgacaacaacggcgccgtgaagttccctcagctgtgcaagttctgcgacgtgaggttcagcacctgcgacaaccagaagtcctgcatgagcaactgcagcatcacaagcatctgcgagaagccccaggaggtgtgtgtggccgtgtggaggaagaacgacgaaaacatcaccctcgagaccgtgtgccatgaccccaagctgccctaccacgacttcatcctggaagacgccgcctcccccaagtgcatcatgaaggagaagaagaagcccggcgagaccttcttcatgtgcagctgcagcagcgacgagtgcaatgacaacatcatctttagcgaggagtacaacaccagcaaccccgacTGATAA SEQ ID NO: 6Polypeptide sequence of the secreted lambda lightchain of anti-PD-L1(mut)/TGFβ Trap, with mutations A31G, D52E, R99YQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTYVEGTGTKVTVLGQPKANPTVTLEPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVT HEGSTVEKTVAPTECSSEQ ID NO: 7 Polypeptide sequence of the secreted heavy chainof anti-PD-L1 (mut)/TGFβ TrapEVQLLESGGGLVQPGGSLRLSCAASGFTESMYMMMWVRQAPGKGLEWVSSIYPSGGITFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFELYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGAGGGGSGGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE YNTSNPD SEQ ID NO: 8Human TGFβRII Isoform A Precursor Polypeptide(NCBI RefSeq Accession No: NP_001020018)MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEEYASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTARYMAPEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK SEQ ID NO: 9Human TGFβRII Isoform B Precursor Polypeptide(NCBI RefSeq Accession No: NP_003233MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEEYASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTARYMAPEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGR SCSEEKIPEDGSLNTTKSEQ ID NO: 10 A Human TGFβRII Isoform B Extracellular Domain PolypeptideIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD SEQ ID NO: 11 (G1y₄Ser)₄Gly linkerGGGGSGGGGSGGGGSGGGGSG SEQ ID NO: 12Polypeptide sequence of the secreted heavy chainvariable region of anti-PD-L1 antibody MPDL3280AEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARR HWPGGFDYWGQGTLVTVSSSEQ ID NO: 13 Polypeptide sequence of the secreted light chainvariable region of anti-PD-L1 antibody MPDL3280Aand the anti-PD-L1 antibody YW243.55S70DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQ GTKVEIKRSEQ ID NO: 14 Polypeptide sequence of the secreted heavy chainvariable region of anti-PD-L1 antibody YW243.55S70EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRH WPGGFDYWGQGTLVTVSA

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientificarticles referred to herein is incorporated by reference for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting the invention described herein. Various structuralelements of the different embodiments and various disclosed method stepsmay be utilized in various combinations and permutations, and all suchvariants are to be considered forms of the invention. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

1. A protein comprising: a) human TGFβRII, or a fragment thereof capableof binding TGFβ; and b) an antibody, or an antigen-binding fragmentthereof, that binds human protein Programmed Death Ligand 1 (PD-L1). 2.A polypeptide comprising: a) at least a variable domain of a heavy chainof an antibody that binds human protein Programmed Death Ligand 1(PD-L1); and b) human TGFβRII, or a fragment thereof capable of bindingTGFβ.
 3. The polypeptide of claim 2, further comprising an amino acidlinker connecting the C-terminus of the variable domain to theN-terminus of the human TGFβRII or fragment thereof.
 4. The polypeptideof claim 3, comprising the amino acid sequence of SEQ ID NO:
 3. 5. Anucleic acid comprising a nucleotide sequence encoding a polypeptideaccording to claim
 2. 6. The nucleic acid of claim 5, further comprisinga second nucleotide sequence encoding at least a variable domain of alight chain of an antibody which, when combined with the polypeptide,forms an antigen-binding site that binds PD-L1.
 7. The nucleic acid ofclaim 6, wherein the second nucleotide sequence encodes the amino acidsequence of SEQ ID NO: 1 (secreted anti-PD-L1 lambda light chain).
 8. Acell comprising the nucleic acid of claim 5 and a second nucleic acidencoding at least a variable domain of a light chain of an antibodywhich, when combined with the polypeptide, forms an antigen-binding sitethat binds PD-L1.
 9. The cell of claim 8, wherein the second nucleicacid encodes the amino acid sequence of SEQ ID NO:
 1. 10. A cellcomprising the nucleic acid of claim
 6. 11. A method of producing aprotein comprising a) TGFβRII, or a fragment thereof capable of bindingTGFβ, and b) an antibody, or an antigen-binding fragment thereof, thatbinds human protein Programmed Death Ligand 1 (PD-L1), the methodcomprising maintaining a cell according to claim 8 under conditionspermitting expression of the protein.
 12. The method of claim 11,further comprising harvesting the protein.
 13. A protein comprising: apolypeptide according to claim 2; and at least a variable domain of alight chain of an antibody which, when combined with the polypeptide,forms an antigen-binding site that binds PD-L1.
 14. The protein of claim13, comprising two polypeptides each having an amino acid sequenceconsisting of the amino acid sequence of SEQ ID NO: 3, and twoadditional polypeptides each having an amino acid sequence consisting ofthe amino acid sequence of SEQ ID NO:
 1. 15-35. (canceled)